Strains of Colletotrichum coffeanum Noack Causing Coffee Berry Disease in Angola and Malawi with Characteristics Different to the Kenya Strain

Strains of Colletotrichum isolated from berries of Coffea arabica from Angola and Malawi were identified as C. coffeanum causing coffee berry disease (CBD). The isolates were highly aggressive to berries of the cultivar ‘Catimor’ and produced high numbers of acervuli and extremely large masses of pink conidia when cultivated in the dark. These strains are apparently different in aggressiveness and morphology to those described in the past from Kenya.     

Molecular analysis of introgressive breeding in coffee (Coffea arabica L.)

Nineteen arabica coffee introgression lines (BC₁F₄) and two accessions derived from a spontaneous interspecific cross (i.e. Timor Hybrid) between Coffea arabica (2n=4x=44) and C. canephora (2n=2x=22) were analysed for the introgression of C. canephora genetic material. The Timor Hybrid-derived genotypes were evaluated by AFLP, using 42 different primer combinations, and compared to 23 accessions of C. arabica and 8 accessions of C. canephora. A total of 1062 polymorphic fragments were scored among the 52 accessions analysed. One hundred and seventy-eight markers consisting of 109 additional bands (i.e. introgressed markers) and 69 missing bands distinguished the group composed of the Timor Hybrid-derived genotypes from the accessions of C. arabica. AFLP therefore seemed to be an extremely efficient technique for DNA marker generation in coffee as well as for the detection of introgression in C. arabica. The genetic diversity observed in the Timor Hybrid-derived genotypes appeared to be approximately double that in C. arabica. Although representing only a small proportion of the genetic diversity available in C. canephora, the Timor Hybrid obviously constitutes a considerable source of genetic diversity for arabica breeding. Analysis of genetic relationships among the Timor Hybrid-derived genotypes suggested that introgression was not restricted to chromosome substitution but also involved chromosome recombinations. Furthermore, the Timor Hybrid-derived genotypes varied considerably in the number of AFLP markers attributable to introgression. In this way, the introgressed markers identified in the analysed arabica coffee introgressed genotypes were estimated to represent from 9% to 29% of the C. canephora genome. Nevertheless, the amount of alien genetic material in the introgression arabica lines remains substantial and should justify the development of adapted breeding strategies. Received: 2 February 1999 / Accepted: 12 May 1999     

Effect of auxins on flower formation in coffee (Coffea arabica L.)

With the aim to reduce the period of flowering and of fruit maturation, we investigated the effect of auxins on flower formation. For these experiments we used young decapitated plants with two plagiotropic branches. Both the auxins, indol-3-ylacetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D), retarded flower formation in coffee, the latter one being more effective. The effects of 2,4-D if applied on only one of the two plagiotropic branches can be observed only in this treated one. Furthermore, the auxins seem to act in coffee plant directly by affecting flower formation and not indirectly by inducing endogenous ethylene production.     

Biotechnological applications for the improvement of coffee (Coffea arabica L.)

Summary The important advances in coffee biotechnological techniques which have been made particularly during the last 10yr could benefit the coffee breeder in practice and open new perspectives for the development of new varieties. The molecular phylogeny of Coffea species has been established using DNA sequence data. The molecular markers have revealed an extremely reduced genetic diversity in Coffea arabica L. in comparison to C. canephora. However, wild accessions collected in the Ethiopian highlands appeared to constitute a valuable gene reservoir. A complete genetic linkage map of C. canephora was reported and additional ones are being constructed, particularly on C. arabica. The integration of Molecular Assisted Selection in coffee breeding promises to drastically increase the efficiency of breeding programs. Economically important genes of the caffeine biosynthetic pathway or genes encoding for seed storage proteins have been isolated. The high performance already achieved in the in vitro propagation process by somatic embryogenesis offers the possibility to mass propagate superior hybrids in different countries of both C. arabica (selected F₁ hybrids) and C. canephora (rootstock variety). Pilot productions by somatic embryogenesis currently permit preparation for commercial application. Somaclonal variation was observed. The percentage of the off-types can vary between 3 and 10% depending on the genotype. Seed cryopreservation enables a routine use for long-term conservation of coffee genetic resources. Transgenic plants have been obtained for the C. arabica and C. canephora cultivated species through Agrobacterium-mediated transformation which constitutes the technique now currently used to transfer directly genes in coffee plants.     

Strains of Colletotrichum coffeanum, the causal agent of coffee berry disease, tolerant to benzimidazole compounds in Kenya

The detection of Colletotrichum coffeanum tolerant to methyl ester of benzimidazole 2-carbamic acid (carbendazim) and a related benzimidazole compound, cypendazole, followed increases in levels of coffee berry disease observed on Coffea arabica in experimental plots sprayed for 2 yr with these compounds. Sporulation by the pathogen on naturally infected berries removed from carbendazim-, cypendazole- or benomyl-sprayed plots was not checked by a further application of 0–05 % (a.i.) of any of the compounds. Nearly all the isolates from these berries were capable of some growth on agar media containing 1000 ppm (a.i.) of either carbendazim or cypendazole. However, only a few could tolerate 1000 ppm of benomyl and the inability of this compound to reduce sporulation on berries infected with tolerant strains was presumably due to its rapid conversion to carbendazim within the host tissue. Less than 1 ppm of carbendazim, cypendazole or benomyl was needed to give 50% inhibition of conidia of the normal strain. Against the most tolerant strains, however, the LD 50 was > 100 ppm of carbendazim and about 30 ppm of benomyl. Whether isolated from unsprayed or benzimidazole-sprayed plots, all isolates of Colletotrichum acutatum, a saprophytic cohabitant of lesions initiated on berries by C. coffeanum, showed the highest degree of tolerance to benzimidazole compounds. No tolerance of either fungus to the ‘conventional’ fungicide captafol was detected.     

Tolerance in Isolates of Colletotrichum coffeanum to Prochloraz-Mn in Kenya

Coffee berry disease, caused by Colletotrichum coffeanum is a serious disease of Coffea arabica in Kenya. Control of this disease is achieved mainly through the use of fungicides which currently include chlorothalonil and prochloraz-Mn (Octave). Fungicide resistance in C. coffeanum to benzimidazoles has been well documented. Isolates of C. coffeanum highly sensitive to prochloraz-Mn have been shown to acquire tolerance to high levels of prochloraz-Mn in culture. These isolates were able to grow and sporulate in malt extract agar (MEA) amended with 250 μg ml^-1 prochloraz-Mn. The prochloraz-Mn tolerant isolates of C. coffeanum showed low level of tolerance to benomyl. The benomyl tolerant isolates of C. coffeanum equally showed low level of tolerance to prochloraz-Mn. The prochloraz-Mn and benomyl tolerant and sensitive isolates were found to be highly pathogenic and induced sporulating lesions on seedlings and berries of coffee cv. SL 28 whichis, very susceptible to C. coffeanum. Mixed inoculation tests using prochloraz-Mn and benomyl tolerant isolates and prochloraz-Mn tolerant and sensitive isolates of C. coffeanum showed that the prochloraz-Mn tolerant isolate was competitive.     

Genetic diversity of forest arabica coffee (Coffea arabica L.) in Ethiopia as revealed by random amplified polymorphic DNA (RAPD) analysis

Genetic diversity within the forest Coffea arabica L. gene pool in Ethiopia has not been extensively examined with molecular markers. In the present study, a total of 75 polymorphic RAPD bands generated by twelve random primers were used to assess genetic diversity among 144 genotypes representing 16 C. arabica populations. The number of polymorphic bands detected with each primer ranged from 2 to 9 with a mean of 6.25 bands per primer. Banding patterns ranged in percentage polymorphism from 37% to 73% with an overall mean of 56% for the populations analyzed. The amount of genetic variation among populations estimated by Shannon-Weaver diversity index was (H = 0.30). The within population and between populations differentiation values were 0.65 and 0.35, respectively. Genetic differentiations within and between zones of sample collection sites were 0.80 and 0.20, respectively. Within population average similarities estimated by simple matching coefficients ranged from 0.72 to 0.85, with an overall average of 0.78. In the cluster analysis that used individual samples as operational taxonomic units, most of the representatives of the same population failed to cluster before they joined members of other populations. Nevertheless, most of the populations were clustered on the basis of their geographic closeness and an east west differentiation was observed at approximately 75% similarity. The results obtained provide information on how to select sites for in situ conservation of C. arabica germplasm.     

Changes in some characteristics between the wild and Al-tolerant coffee (Coffea arabica L.) cell line

An aluminium (Al)-tolerant cell line (LAMt) of coffee (Coffea arabica L.) was obtained from a cell suspension culture and biochemically and molecularly characterized in an MS medium at half ionic strength and low pH. LAMt grew 30% more than the control line (susceptible to Al) in the presence of different concentrations of Al, showed a lower free Al concentration in the medium and had higher phospholipase C specific activity (80%). Membrane integrity of the LAMt was 50% greater than the control line when both were incubated in the presence of different Al concentrations (measured by Evans Blue uptake). Finally, the use of microsatellite primers revealed no difference in the DNA pattern of both cell lines.     

Analysis of genetic structure in a sample of coffee (Coffea arabica L.) using fluorescent SSR markers

The knowledge of population structure is important to determine the degree of linkage disequilibrium, which allows the selection of genotypes for association mapping. Using 47 SSR markers, the genetic variability and population structure of 68 accessions of C. arabica (wild and cultivated) and of three diploid species used as reference were evaluated. The analysis was done with the distance method and the structure model. The structure analysis inferred nine subpopulations (k = 9), for which the greatest values of probability were obtained. Three of the groups corresponded to the three diploid species as expected. There were six groups identified within C. arabica. The genetic subdivisions within C. arabica were based on geographical origin, degree of domestication, and dispersal history of coffee. One group consisted entirely of cultivated genotypes, where intense population bottleneck were associated with a founder effect. This was the most homogeneous group, as demonstrated by the reduced distance between cultivars in the dendrogram. Three of the cultivated genotypes, originating from Sudan, were separated into an independent group, presumably due to selective adaptation to a different set of environmental conditions. Another group consisted of genotypes of the type “ennarea” that were grown and cultivated in isolation on the shores of the Tana lake. The semi-wild genotypes clustered into three different groups. This type of analysis provides a strong evidence of population structure in C. arabica. Based on these findings, it is possible to better identify a balanced sample of diverse plants in germplasm.     

Inter-simple sequence repeat (ISSR) variation in forest coffee trees (Coffea arabica L.) populations from Ethiopia

Genetic variation of forest coffee trees (Coffea arabica L.) from four regions of Ethiopia was investigated using inter-simple sequence repeat (ISSR) markers. A total of 160 individuals representing 16 populations were sampled. Eleven ISSR primers amplified a total of 123 fragments of which 31 fragments (25%) were polymorphic. Estimate of total gene diversity (H _T), and the coefficient of genetic differentiation (G _ST) were 0.37 and 0.81, respectively. This indicates that most of the variability is between populations than within populations. The partitioning of genetic variation into within and between populations based on Shannon’s information index also revealed more differentiation between populations (0.80) than within populations (0.20). In the phenogram most of the coffee tree samples were clustered on the basis of their regions of origin but failed to cluster according to their respective populations, which could be attributed to the presence of substantial gene flow between adjacent populations in each region assisted by man in the process of transplantation or by wild animals such as monkeys, which eat the berries and defecate the seeds elsewhere. On the other hand, the inter-regional clustering of some coffee tree samples from Bale and Jimma regions could be due to the transport of coffee seeds across regions and their subsequent planting. Although ISSR markers detected lower polymorphic loci than previously reported results with random amplified polymorphic DNA (RAPD) markers on the same materials, it can be used as an alternative method for molecular characterization of C. arabica populations. The results may provide information to select sites for in situ conservation.     

Biotransformation of theobromine to caffeine in suspension and polyurethane foam immobilized coffee (Coffea arabica L.) cells

Coffee (Coffea arabica L.) cells are capable of biotransforming theobromine to caffeine. In suspension culture of B2K medium, which is the production medium for caffeine, biotransformation was also more efficient than in DK medium. More caffeine was finally produced than calculated based on theobromine added to the medium. On the other hand, the efficiency of the biotransformation using immobilized cells in reticulate polyurethane foam cubes as a matrix varied with the phases. The biotransformation tended to be efficient under conditions which allowed the coffee cells to vigorously produce caffeine de novo.This paper is Part 75 in the series of Studies on Plant Tissue Cultures. For Part 74, see Kawaguchi K, Hirotani M, Furuya T, (1991) Phytochemistry, in press.     

Modification of the culture medium to produce aluminum toxicity in cell suspensions of coffee (Coffea arabica L.)

Coffee (Coffea arabica) plants are usually grown in soils containing high levels of organic materials. Under these conditions, aluminum (Al) is toxic because of the acidic nature of the soils. Al is the most abundant metal found in the earth's crust and occurs in a number of different forms in soil. In acid soils, Al toxicity is a global problem that limits crop productivity. A major problem in obtaining cellular lines displaying Al tolerance in culture is the composition of the medium. In the experiments presented here, we modified the composition of the culture medium for a C. arabica cell line to produce Al toxicity. Murashige-Skoog media was used, complete (MS) and half ionic strength (MSHIS), at either pH 5.8 or 4.3. We found that MSHIS and pH 4.3 provided the optimal conditions to obtain Al toxicity as measured by the ability to grow in a range of Al concentrations (25-1,000 µM). The lethal dose (LD₅₀) under these conditions was 25 µM. The concentrations of free Al in the culture medium were corroborated by the fluorescent compound Morin. Al was found to enter the cell after 30 min, and the signal was then retained for up to 2 h.     

Specific PCR-based detection of Phomopsis heveicola the cause of leaf blight of Coffee (Coffea arabica L.) in China

Coffee (Coffea arabica L.) is currently grown in many tropical and subtropical areas countries and is a major traded commodity for the developing world. Coffee leaf blight, caused by Phomopsis heveicola, is one of the most important fungal diseases dangerous to coffee crops in China. This study aimed to develop a PCR-based diagnostic method for detecting P. heveicola in planta. Specific primers (CPHF/CPHR) were designed based on sequence data of region of internal transcribed spacer (ITS1 and ITS4) of P. heveicola. The efficiency and specificity of CPHF/CPHR were established by PCR analysis of DNA from P. heveicola strains isolated from China and fungal isolates of other genera. A single amplification product of 318 bp was detected from DNA P. heveicola isolates. No amplification product was observed with any of the other fungal isolates tested. The specific primers designed and employed in PCR detected P. heveicola up to 3 pg from DNA isolated. This is the first report on the development of a species-specific PCR assay for identification and detection of P. heveicola. Thus, the PCR-based assay developed was very specific, rapid and sensitive tool for the detection of pathogen P. heveicola.     

Effect of pore size and shape on the immobilization of coffee (Coffea arabica L.) cells in porous matrices

Summary The pore size and shape of porous matrices were evaluated as to their effect on the immobilization efficiency in cultured coffee (Coffea arabica L.)/cells. A hydrophilic porous matrix (13–20 pores/25 mm) and reticulate polyurethane foam (30 pores/25 mm) indicated more efficient immobilization than the others, in small cubes (1 cm³ × 9) and a strip (1 × 1 × 9 cm³) at the end of the fourth subculture. Among the large cubes (9 cm³), the reticulate one with the largest pore size (13 pores/25 mm) was the most advantageous for immobilization. In the strip-shaped matrices (1 × 1 × 9 cm³), immobilization was the most efficient in spite of its lower surface area as compared to the small cubes, except for those with the largest pore size. The strip-shaped foams, which were fixed on the inside of the flask against shaking, were effective for immobilization. Finally, strips (30 pores/25 mm) with slits to increase the surface area of the foam immobilized the largest amount of cells at the end of the fourth subculture. Caffeine production was not changed by diffenences in pore size.This paper is Part 76 in the series of Studies on Plant Tissue Cultures. For Part 75, see Furuya T., Orihara Y., Koge K. (1991) Plant Cell Rep 9:659–662 Offprint requests to: T. Furuya