Cytogenomic characterization of Colletotrichum kahawae,the causal agent of coffee berry disease,reveals diversity in minichromosome profiles and genome size expansion

Colletotrichum kahawae is an emerging fungal pathogen, which has recently undergone a speciation process from a generalistic ‘C. gloeosporioides species complex' background by acquiring the unique capacity to infect green coffee berries, thus causing coffee berry disease. This is a severe and widespread disease in Africa and an imminent threat to Arabica coffee cultivation in Asia and America, if the pathogen enters those continents. Genetic diversity within C. kahawae is low but notorious differences in pathogen aggressiveness have been described. This work characterized two cytogenomic traits (genome size and minichromosome profiles) of a collection of C. kahawae isolates, representing the breadth of its genetic diversity and distinct aggressiveness classes, along with closely related taxa. The results obtained constitute the first flow cytometry‐based genome size estimation in the genus Colletotrichum and show a c. 8 Mb genome size expansion between C. kahawae (79·5 Mb on average) and its closest relatives (71·3 Mb), corroborating evidence indicating that C. kahawae (i.e. the coffee berry disease pathogens) should remain as a distinct species. Results have also shown the presence of two to five minichromosomes in C. kahawae, suggesting a positive relationship between the number of minichromosomes and the level of aggressiveness of the different isolates analysed, while no correlation could be established between aggressiveness and whole genome size. Overall, these results may be the basis for the identification of pathogenicity/aggressiveness‐related factors in such minichromosomes, and may provide clues to the characterization of specific markers for aggressiveness classes.     

Identification of <Emphasis Type="Italic">Colletotrichum</Emphasis> species associated with anthracnose disease of coffee in Vietnam

Twenty-three isolates of Colletotrichum gloeosporioides, five isolates of C. acutatum, two isolates of C. capsici and six isolates of C. boninense associated with anthracnose disease on coffee (Coffea spp.) in Vietnam were identified based on morphology and DNA analysis. Phylogenetic analysis of DNA sequences from the internal transcribed spacer region of nuclear rDNA and a portion of mitochondrial small subunit rRNA were concordant and allowed good separation of the taxa. We found several Colletotrichum isolates of unknown species and their taxonomic position remains unresolved. The majority of Vietnamese isolates belonged to C. gloeosporioides and they grouped together with the coffee berry disease (CBD) fungus, C. kahawae. However, C. kahawae could be distinguished from the Vietnamese C. gloeosporioides isolates based on ammonium tartrate utilization, growth rate and pathogenicity. C. gloeosporioides isolates were more pathogenic on detached green berries than isolates of the other species, i.e. C. acutatum, C capsici and C. boninense. Some of the C. gloeosporioides isolates produced slightly sunken lesions on green berries resembling CBD symptoms but it did not destroy the bean. We did not find any evidence of the presence of C. kahawae in Vietnam.     

Proteomic analysis of Colletotrichum kahawae-resistant and susceptible coffee fruit pericarps

Coffee berry disease (CBD) is caused by the fungus Colletotrichum kahawae and is restricted to the African continent, where it generates losses of up to 80 % of coffee production. Weather conditions in certain growing areas at high altitudes in Colombia appear to be very favourable for the development of this disease. Certain genotypes of Coffee arabica are resistant to this pathogen, such as the Timor Hybrid and some Ethiopian accessions. It is important to identify the proteins in these coffee genotypes that are associated with resistance to this fungus. Therefore, we compared the proteomes of two genotypes that are resistant to different isolates of C. kahawae with the proteome of the susceptible coffee genotype Caturra. We optimized the methodology applied for the extraction, cleaning and purification of proteins from the green fruit pericarp at 150 to 170 days after flowering. Through two-dimensional differential gel electrophoresis, proteomic map images were obtained for the resistant and susceptible genotypes. Fifty-two protein spots that were significantly different between the resistant and susceptible genotypes were detected. These protein spots were isolated and sequenced via mass spectrometry. The sequence analysis identified 14 proteins in the Timor Hybrid and 14 in CCC1147 that were associated with resistance and pathogen defence.     

Identification of molecular markers linked to a gene conferring resistance to coffee berry disease (Colletotrichum kahawae) in Coffea arabica

Coffee berry disease (CBD) caused by Colletotrichum kahawae is a major constraint to Arabica coffee (Coffea arabica) production in Africa. One source of resistance to the disease is a natural interspecific hybrid between C. arabica and C. canephora and its derivatives. This study is aimed at deciphering the genetic basis of the host resistance and identification of molecular markers associated with it. CBD is a mature stage disease and in the absence of a mature mapping population, early detection of disease reaction phenotypes of mapping individuals is required. Two F₂ populations from crosses of cv. Catimor (resistant) and cv. SL28 (susceptible) were screened for resistance by a two step procedure. First, half of each population was screened 6 weeks after germination by inoculating hypocotyls with the pathogen. The surviving seedlings (G1) were considered to be resistant and were raised in a nursery together with the other unscreened halves (G2). Secondly, after one year, all the seedlings (G1 + G2) were screened by inoculation. Analysis of 57 microsatellites and 31 AFLP markers in 56 and 95 seedlings from G1 and G2, respectively, were performed. Eight AFLP and two microsatellites markers linked tightly to the resistant phenotype were identified and mapped to one unique chromosomal fragment introgressed from C. canephora. The gene conferring the resistance was localized within an 11 cM segment. It is concluded that the locus carries a major resistance gene designated Ck‐1, which is likely to be synonymous to the T gene described in previous studies.     

Aggressiveness of eight <Emphasis Type="Italic">Didymella rabiei</Emphasis> isolates from domesticated and wild chickpea native to Turkey and Israel,a case study

Ascochyta blight, caused by Didymella rabiei, affects both domesticated chickpea and its congeneric wild relatives. The aim of this study was to compare the aggressiveness of D. rabiei isolates from wild and domesticated Cicer spp. in Turkey and Israel on wild and domesticated hosts from both countries. A total of eight isolates of D. rabiei sampled from C. pinnatifidum, C. judaicum and C. arietinum in Turkey and Israel was tested on two domesticated chickpea cultivars and two wild Cicer accessions from Turkey and Israel. Using cross-inoculation experiments, we compared pathogen aggressiveness across the different pathogen and host origin combinations. Two measures of aggressiveness were used, incubation period and relative area under the disease progress curve. The eight tested isolates infected all of the host plants, but were more aggressive on their original hosts with one exception; Turkish domesticated isolates were less aggressive on their domesticated host in comparison to the aggressiveness of Israeli domesticated isolates on Turkish domesticated chickpea. C. judaicum plants were highly resistant against all of the isolates from different origins except for their own isolates. Regardless of the country of origin, the wild isolates were highly aggressive on domesticated chickpea while the domesticated isolates were less aggressive on the wild hosts compared with the wild isolates. These results suggest that the aggressiveness pattern of D. rabiei on different hosts could have been shaped by adaptation to the distinct ecological niches of wild vs. domesticated chickpea.     

Distinguishing characteristics and vegetative compatibility of Colletotrichum kahawe in comparison with other related species from coffee

On the basis of pathogenicity tests on green berries or hypocotyls of coffee and by morphological and biochemical characteristics in culture, 31 isolates of Colletotrichum were classified into C. kahawe (24 isolates), C. gloeosporioides (six isolates) or C. acutatum (one isolate). Within these groups of isolates, vegetative compatibility groups (VCGs) were determined by complementation tests with mutants in the nitrate assimilation pathway. There were distinct incompatibility barriers between the three species. Among the C. gloeosporioides group, the three isolates tested were self-compatible but incompatible with each other. Within C. kahawe , 18 isolates were self-compatible and only one main VCG was detected. However, partial compatibility in C. kahawe was also indicated by variation in the intensity of heterokaryon formation between different pairs of isolates and between different types of mutant. The existence of only one VCG in C. kahawe is consistent with the low level of variation found in previous work on DNA polymorphism.     

Effect of cultural practices on the development of arabica coffee berry disease,caused by <Emphasis Type="Italic">Colletotrichum kahawae</Emphasis>

In the high altitude regions of Africa, coffee berry disease (CBD), caused by Colletotrichum kahawae, is the main constraint for arabica coffee (Coffea arabica) production. However, certain agricultural practices can reduce losses caused by the disease and thereby promote optimum production. On small family farms in Cameroon, mixed cropping with fruit trees, intercropping with food crops and maintenance pruning of coffee trees are very widespread agricultural practices that can affect CBD epidemics. Consequently, an epidemiological study was conducted to assess how cultural practices affected the disease in an arabica coffee smallholding in Cameroon. The disease was monitored on a weekly basis over four successive years (2002–2005) on coffee trees in diverse cultural situations. Cultural practices likely to reduce losses due to CBD were identified. The infection rate was significantly lower on coffee trees grown intensively than on coffee trees grown in the traditional manner. Coffee trees located under the shade of fruit trees were significantly less attacked than those located in full sunlight. In addition, berries on the leafless parts of branches, near the main trunk of the coffee tree, were less infected than those on leafy sections. These results show that maintenance pruning, removal of mummified berries, and mixed cropping with shade plants are cultural practices which create environmental conditions that limit CBD development.     

Genetic diversity and structure of Hemileia vastatrix populations on Coffea spp.

Coffee leaf rust is the most limiting disease for coffee cultivation in Brazil. Despite its importance, relatively little is known about the genetic diversity of Hemileia vastatrix, the rust causal agent. In this work, the DNA from 112 monopustule isolates from different geographic locations and coffee genotypes were analysed by amplified fragment length polymorphisms (AFLP). The objectives were to assess the influence of the host and geographic origin on the diversity and population differentiation in H. vastatrix. The fungal population showed a low level of genotypic diversity. Gene diversity (h) was 0·027 and the hypothesis of random mating in the total population was rejected, but evidence for recombination was found for two subpopulations (São Paulo and Paraná). The analysis of molecular variance revealed that 90% of the genetic distribution of the pathogen occurs among isolates within the subpopulation (states or host of origin). There was no correlation between geographic and genetic distance (r = ?0·024, P = 0·74), which together with the high number of migrants and the low degree of differentiation in populations of H. vastatrix, is consistent with the fact that the inoculum is probably easily dispersed by wind over long distances, allowing dispersal of the pathogen among coffee growing areas in Brazil. Therefore, it is difficult to predict the durability of resistant sources to coffee rust. The recommendation for the breeding programmes is thus to incorporate multigenic resistance as a control strategy.     

Characteristics and distribution of Colletotrichum species in coffee plantations in Hainan,China

Anthracnose caused by Colletotrichum species is a serious disease on a range of economically important hosts. To determine the Colletotrichum species in coffee plantations in Hainan, China, 55 isolates were obtained from Coffea arabica (arabica) and C. canephora var. robusta (robusta) in five counties. Initially, partial sequences of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to measure fungal genetic diversity. Then a subset of 23 isolates was selected to represent the range of genetic diversity, varieties and geographic origin for further multilocus phylogenetic analyses. These isolates belong to eight known Colletotrichum species from three Colletotrichum species complexes, including gloeosporioides (C. endophytica, C. fructicola, C. ledongense, C. siamense and C. tropicale), boninense (C. karstii), gigasporum (C. gigasporum), and one singleton species (C. brevisporum). Of these, C. siamense was isolated in all sampled counties and C. fructicola was identified in three counties. The other six species were isolated only in one or two counties. Only C. siamense and C. fructicola were isolated from arabica, whereas all eight species were isolated from robusta. Occurrence of C. brevisporum, C. endophytica, C. ledongense and C. tropicale in coffee has not been reported previously. Pathogenicity tests showed that all eight species were pathogenic to coffee leaves and fruit. In vitro tests showed that Colletotrichum isolates from coffee in Hainan were most sensitive to prochloraz, less sensitive to carbendazim, propiconazole and difenoconazole, and least sensitive to myclobutanil.     

Species of the Colletotrichum gloeosporioides and C. boninense complexes associated with olive anthracnose

The taxonomic status of Colletotrichum gloeosporioides sensu lato (s.l.) associated with olive anthracnose is still undetermined and the pathogenic ability of this species complex is controversial. In the present study, isolates obtained from olive and provisionally identified as C. gloeosporioides s.l. on the basis of morphological and cultural features were reclassified using ITS and TUB2 as DNA barcode markers and referred to seven distinct species, recently separated within C. gloeosporioides (C. aenigma, C. gloeosporioides sensu stricto (s.s.), C. kahawae, C. queenslandicum, C. siamense and C. theobromicola) and C. boninense (C. karstii) species complexes. Furthermore, isolates of C. kahawae were ascribed to the subspecies ciggaro by analysing the GS gene. A single isolate, not in either of these two species complexes, was not identified at the species level. In pathogenicity tests on detached olive drupes some of these species, including C. aenigma, C. kahawae subsp. ciggaro, C. queenslandicum, C. siamense and C. karstii, were shown to be weakly pathogenic. Moreover, they were found very sporadically on olive. In contrast, some isolates of C. gloeosporioides s.s. and isolates of C. theobromicola proved to be virulent on both green and ripening olives. This study gives a better insight into both the aetiology and the epidemiology of olive anthracnose and might have implications for biosecurity and quarantine because C. theobromicola has never been reported in major European olive‐producing countries.     

Variability in aggressiveness of Quambalaria pitereka isolates

Quambalaria shoot blight, caused by the fungal pathogen Quambalaria pitereka, is a serious disease of eucalypt plantations in Australia. The aggressiveness of four Q. pitereka isolates was compared on a range of host genera, species, provenances and clones. Isolates differed substantially in their aggressiveness, with two consistently showing higher levels of aggressiveness based on incidence and severity of disease and lesion size. Isolates derived from Corymbia citriodora subsp. variegata (Ccv) and C. torelliana were shown to have a relatively restricted host range, with lesions but no sporulation found on Eucalyptus species, Angophora species other than A. costata and Corymbia species other than Ccv, the host of origin. The level of aggressiveness toward the different provenances of spotted gum and C. torelliana varied between isolates and there was evidence of some isolate × host interaction within provenances of Ccv. The two methods of inoculation used in this study, spray and spot inoculation, gave similar results. However, the fact that the spot inoculation method was labour‐intensive was a disadvantage limiting the numbers of isolates and hosts that can be tested.     

Genetic diversity and aggressiveness of Erwinia psidii on Eucalyptus spp. in Brazil

The genetic variability and aggressiveness of Brazilian Erwinia psidii isolates from Eucalyptus spp. was studied and compared with reference isolates from guava (Psidium guajava). Repetitive element sequence (rep)-based PCR markers of 101 isolates from Eucalyptus spp. and five from guava showed that the populations of E. psidii displayed a relatively low genetic variability. No correlation of genetic clustering based on rep-PCR analysis with geographic origin or host of origin was observed, indicating that genome rearrangements associated with adaptation to a particular host were not detected by these molecular markers. A higher genotypic richness was detected in the Mato Grosso do Sul population, probably reflecting a pathogen dissemination associated with the recent expansion in eucalypt plantations. Wilcoxon and ANOVA tests of disease severity data indicated differences in aggressiveness among isolates and an isolate × clone interaction. The area under the disease progress curve (AUDPC) and disease severity for some isolates were significantly different between two susceptible clones tested. Notably, isolate LPF681 from guava was not able to cause disease on a susceptible Eucalyptus urophylla clone, suggesting that some co-evolution between pathogen and host has taken place. The variability in aggressiveness and virulence among isolates of E. psidii observed in this study will be important for the establishment of appropriate screening approaches to select for disease resistance.     

Role of rainfall in the development of coffee berry disease in Coffea arabica caused by Colletotrichum kahawae,in Cameroon

The development of coffee berry disease (CBD) epidemics (caused by Colletotrichum kahawae) in Cameroon was monitored over two successive years (2004 and 2005) on coffee trees protected from rainfall by transparent plastic sheets and on unprotected control trees. This work was done to assess how rain affected disease development when it did not fall directly onto the coffee trees and to determine the influence of primary inoculum on the severity of CBD. Weekly observations over the 2 years showed that there were 1·1% diseased berries on coffee trees completely protected from rainfall, compared with 45% diseased berries on unprotected coffee trees. Disease severity on unprotected trees during the 2 years of the experiment was estimated at 53% diseased berries, compared with 27% on trees only protected in the first year. These results confirmed rainfall as one of the key physical factors in the development of Arabica CBD. They also provided evidence of a subsequent effect of protecting coffee trees from rainfall in 2004 on the severity of CBD in 2005. This suggested some practices that might lead to very effective cultural control of CBD in regions where severe epidemics of the disease occur.     

Variation among Colletotrichum gloeosporioides isolates from infected coffee berries at different locations in Vietnam

Isolates of Colletotrichum gloeosporioides associated with anthracnose disease on coffee berries in Vietnam were characterized by morphological and molecular methods. Random amplified polymorphic DNA (RAPD) and microsatellite-primered PCR (MP-PCR) analyses were employed to investigate the genetic variation among 38 and 51 isolates of C. gloeosporioides , respectively. According to both methods, the isolates mainly grouped in accordance with geographical origins. Higher genetic variation ( H  = 0·312 and 0·335) in the northern population of C. gloeosporioides than in the southern population ( H  = 0·261 and 0·186), according to the RAPD and MP-PCR markers, respectively, was indicative of a difference between the northern and southern populations. Moderate gene differentiation ( G st = 0·1) between populations from the north and the south was found. However, there was no differentiation between locations within the northern or southern populations, indicating significant gene flow. A four-gamete test indicated a high level of recombination, particularly in the south. The geographic differences may be explained by different histories of coffee cultivation in different parts of Vietnam. The symptoms caused by the Vietnamese isolates on both hypocotyls and green berries were less severe than symptoms caused by the reference CBD (coffee berry disease; Colletotrichum kahawae ) isolates originating from Africa.     

Transmission of <Emphasis Type="Italic">Colletotrichum coccodes via</Emphasis> tomato seeds

Anthracnose of tomato caused by Colletotrichum coccodes is a devastating disease of ripe fruits. This pathogen may also infect tomato roots, stems and leaves. In the present study, C. coccodes is shown to be capable of contaminating seeds collected from artificially inoculated tomato fruits. Seedlings germinating from these infected seeds exhibited disease symptoms and therefore may transmit the pathogen to the next crop. The proportion of infected seeds ranged between 20% and 63% in all C. coccodes isolates tested and correlated with the aggressiveness of the isolates to tomato fruits. Fungicidal treatment of the collected seeds reduced, but did not eliminate, seed infection. A transgenic C. coccodes isolate expressing green fluorescent protein was used to visualize the pathogen. Mycelium was observed both on surfaces of the seed coat and within 1% of the embryos.     

Genetic diversity and aggressiveness of Calonectria pteridis in Eucalyptus spp.

Calonectria leaf blight, caused by Calonectria pteridis, is currently one of the main foliar diseases in eucalypt plantations in Brazil. In warm and high rainfall regions, the disease can be a limiting factor for eucalypt production when planting susceptible genotypes. The most effective method for controlling this disease in the field is the use of resistant genotypes, which requires knowledge of the genetic variability and aggressiveness of the pathogen population for effective deployment of plant resistance. This work evaluated the genetic diversity and aggressiveness of C. pteridis populations obtained from infected eucalypt plants in Monte Dourado (Pará state) and Imperatriz (Maranhão state), Brazil. To study the genetic diversity, 16 ISSR primers were tested, five of which amplified polymorphic, reproducible and informative bands. Thirty-one closely related genotypes were identified from 84 isolates studied, indicating that the population has a low genetic diversity. The aggressiveness of seven isolates, selected according to geographic origin and their clustering in the ISSR-based dendogram, was determined by inoculation of a hybrid Eucalyptus grandis × E. urophylla clone under controlled conditions. Disease severity was assessed by both measuring the percentage of plant defoliation and assigning a score according to a diagrammatic scale of symptoms. A high correlation between the two evaluation methods was observed, which revealed significant differences in aggressiveness among the isolates. The diagrammatic scale is recommended for disease evaluation because results are obtained much faster, before the occurrence of severe defoliation. No correlation between clustering in the ISSR-based phylogenetic analysis and aggressiveness was observed.     

Spatiotemporal distribution of Ascochyta pinodes and Ascochyta pinodella during the winter growing season in France

Ascochyta blight of pea is caused by four related fungi, Ascochyta pisi, Phoma koolunga, Ascochyta pinodes and Ascochyta pinodella. The latter two taxa appear to be much more common and economically significant worldwide but the relative impact of each fungus on ascochyta blight epidemics is not well understood. To study the spatiotemporal distribution of A. pinodes and A. pinodella infecting pea in France, 368 isolates were sampled monthly, from February to May, at three locations (Rennes, Boigneville and Dijon) and molecular markers were used to genotype isolates. The aggressiveness of isolates from the fourth sampling date was estimated using a detached leaf assay on the winter cultivar Enduro. Disease was low during the sampling period as climatic conditions were generally not conducive to disease development (cold temperature, low rainfall). Population genetic analysis showed that 99% of the observed variation could be attributed to variation within populations compared to only 1% among populations. Both species were observed in each location, although A. pinodella was observed at a lower frequency (6–32%). Moreover, results showed that both species could develop on different nodes of the plant. Significant differences in aggressiveness were observed between species and among isolates within species with A. pinodes isolates being significantly more aggressive on average than A.  pinodella isolates. These results emphasize the necessity to study the components of disease complexes in order to understand the impact of pathogen species interactions on disease and yield reduction as well as the dynamics of disease epidemics during the cropping season.     

Temperature adaptation in Australasian populations of Puccinia striiformis f. sp. tritici

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the major fungal pathogens of wheat. A new pathotype was introduced to Australia in 2002 and several derivative pathotypes were detected in subsequent seasons. It has been suggested that the severity of stripe rust outbreaks in Australia since 2002 could be as a result of traits other than virulence in the pathogen population. This study was conducted to investigate the hypothesis that the stripe rust pathogen population dominant in Australia since 2002 was better adapted to warm temperature conditions compared to previous pathogen populations. Sixteen pathotypes were selected to examine the influence of two contrasting temperature regimes during the 24 h incubation (10°C and 15°C) and the subsequent post‐inoculation (17°C and 23°C) periods on latent period and infection efficiency on four susceptible wheat cultivars. In addition, the effect of two contrasting incubation temperatures on urediniospore germination was examined. The results indicated that pathotypes of P. striiformis f. sp. tritici detected after 2002 did not show evidence of adaptation to high temperatures, which suggests that other factors contributed to the observed increased aggressiveness.     

Evidence of cork barrier formation as a resistance mechanism to berry disease (Colletotrichum coffeanum) in arabica coffee

The histology of resistance to coffee berry disease (CBD) was studied in artificially inoculated berries and hypocotyls of 6-week old seedlings of a number of varieties ofCoffea arabica L. In resistant varieties a phellogen was quickly formed some cell layers below the site of infection and progress of the fungal invasion was effectively blocked by a complete barrier of suberized cells. Such barriers were absent or incompletely developed in CBD susceptible varieties. A highly significant correlation (r=0.87) was found between the frequencies of complete barrier formation in berries and in hypocotyls of young seedlings, while both were also highly correlated to observed mature plant resistance (r>-0.93). Resistance to CBD in arabica coffee may to an important extent be based on the formation of cork barriers. These cork barriers confine the pathogen to the small volume of tissue external to the barrier so that its growth is severely restricted. Such a resistance mechanism is likely to be stable (race-nonspecific). The almost identical response to infection observed in berries and hypocotyls provides further evidence that plants with resistance to CBD can be reliably preselected by the hypocotyl inoculation test.