Characterizing plasma membrane H+-ATPase in two varieties of coffee leaf (Coffea arabica L.) and its interaction with an elicitor fraction from the orange rust fungus (H. vastatrix Berk and Br.) race II.

Early intercellular signaling in Coffea arabica L.-Hemileia vastatrix host-pathogen interaction was studied, using inside-out plasma membrane from two varieties of coffee leaf and a fungal fraction to determine the plant's biochemical responses. Microsomal pellets (100,000 x g) from the susceptible (Caturra) and resistant (Colombia) coffee leaf varieties were purified by partitioning in two-polymer DEX (6.3% w/w) and PEG (6.3% w/w) system aqueous phase. Fungal material was obtained from orange rust Hemileia vastatrix Berk and Br. race II urediospore germ tubes. Plasma membrane vesicles were preferentially localized to PEG phase, as indicated by its enzyme marker distribution. Both H(+)-ATPase activities displayed similar kinetic and biochemical characteristics, comparable to those described for P-type ATPases. Several enzymes may play pivotal roles in plants regarding early interaction with fungal elicitors. Studies of fungal fractions' effects on H(+)-ATPase and both varieties' proton pumping activities were thus carried out. Concentration as low as 0.1 Gluc eq. ml(-1) fungal fraction induced specific inhibition of H(+)-ATPase and the resistant variety's proton pumping activities. The present work describes characterizing the H(+)-ATPase plasma membrane from two Coffea arabica L. varieties (Caturra and Colombia) for the first time and the race specific inhibitory effect of a crude fungal fraction on both H(+)-ATPase and the resistant variety's proton pumping activities.     

Emergent spatial structure and pathogen epidemics: the influence of management and stochasticity in agroecosystems

Organisms susceptible to disease, from humans to crops, inevitably have spatial geometry that influence disease dynamics. Understanding how spatial structure emerges through time in ecological systems and how that structure influences disease dynamics is of practical importance for natural and human management systems. Here we use the perennial crop, coffee, Coffea arabica, along with its pathogen, the coffee leaf rust, Hemileia vastatrix, as a model system to understand how spatial structure is created in agroecosystems and its subsequent influence on the dynamics of the system. Here, we create a simple null model of the socio-ecological process of death and stochastic replanting of coffee plants on a plot. We then use spatial networks to quantify the spatial structures and make comparisons of our stochastic null model to empirically observed spatial distributions of coffee. We then present a simple model of pathogen spread on spatial networks across a range of spatial geometries emerging from our null model and show how both local and regional management of agroecosystems interact with space and time to alter disease dynamics. Our results suggest that our null model of evolving spatial structure can capture many critical features of how the spatial arrangement of plants changes through time in coffee agroecosystems. Additionally, we find small changes in management factors that can influence the scale of pathogen transmission, such as shade tree removal, and result in a rapid transition to epidemics with lattice-like spatial arrangements but not with irregular planting geometries. The results presented here may have practical implications for farmers in Latin America who are in the process of replanting and overhauling management of their coffee farms in response to a coffee leaf rust epidemic in 2013. We suggest that shade reduction in conjunction with more lattice-like planting schemes may result in coffee being more prone to epidemic-like dynamics of the coffee leaf rust in the future.     

Partial resistance to leaf rust (Hemileia vastatrix) in coffee (Coffea arabica L.): genetic analysis and molecular characterization of putative candidate genes

One of the major production limiting diseases in coffee is the orange leaf rust caused by the fungus Hemileia vastatrix (Berkeley and Broome). Little is known about the inheritance and genetic determinism of partial resistance in coffee (C. arabica L.) to H. vastatrix. This information would be useful to breed durable resistant cultivars efficiently. In this report, a genetic analysis of partial resistance to leaf rust in Coffea arabica was performed using nine segregating progenies from a cross between the susceptible variety Caturra and the resistant introgressed line DI200. Evolution of partial resistance was evaluated under field conditions by measuring rust incidence (RI) and defoliation (DEF) in two separate regions of productive branches per tree and during four successive years (2003–2006). Genetic components of rust resistance were estimated using the Means and Variance Generation Method, under an additive-dominant model. The most important genetic effect was the additive one, while resistance heritability estimates ranged from 73 to 53% for broad and narrow sense heritabilities, respectively. Genetic estimates for the number of segregating genes showed that at least five independent genes or genetic regions are implicated in the partial resistance to rust. We further analyzed the presence of resistance (RGC) and defense (DGC) gene candidates in the resistant and susceptible parents by using a degenerated-primer PCR approach. A total of 40 different genomic coffee sequences were isolated exhibiting strong similarity to known RGC or DGC homologous. Phylogenetic analysis clustered these sequences into nine families. One family exhibited the TIR protein element, representing the first TIR class proteins identified in coffee. While genetic analysis suggest a predictable success in the processes to improve the selection of resistant lines for future varieties with durable resistance, the molecular characterization of candidate genes represent a primary approach towards the identification of mechanisms involved in partial resistance to coffee leaf rust.     

Impact of the Coffea canephora gene introgression on beverage quality of <Emphasis Type="Italic">C. arabica</Emphasis>

Lines of Coffea arabica derived from the Timor Hybrid (hybrid between C. arabica and C. canephora) are resistant to coffee leaf rust (Hemileia vastatrix) and to the nematode Meloidogyne exigua. The introgression of C. canephora resistance genes is suspected of causing a drop in beverage quality. Coffee samples from pure lines, compared in a Trial 1, and from F1 hybrids and parental lines from a half-diallel trial in a Trial 2, were studied for beverage quality, chemical composition and amount of introgressed genetic material. Chemical analyses (caffeine, chlorogenic acids, fat, trigonelline, sucrose) were carried out with near-infrared spectrometry by reflectance of green coffee. The number of amplified fragment length polymorphic (AFLP) markers introgressed from the Timor Hybrid varied from 1 to 37 for the lines studied. There were significant differences between lines for all of the biochemical compounds analysed and for the acidity and the overall standard of the beverage. Two lines (T17927, T17924) were significantly poorer than the controls for sucrose and beverage acidity. T17924 also had more chlorogenic acids and was poorer for the overall standard. However, two highly introgressed lines, T17934 and T17931 (25 and 30 AFLP markers, respectively), did not differ from the non-introgressed controls. There were no correlations between the number of AFLP markers and the chemical contents or beverage attributes. Significant correlations were found between the performance of the parents and their general combining ability for beverage quality. It was concluded that it should be possible to find lines with both the desired resistance genes and good beverage quality. Selection can avoid accompanying the introgression of resistance genes with a drop in beverage quality.     

The Potential Use of a Silicon Source as a Component of an Ecological Management of Coffee Plants

Coffee is one of the most important agricultural export commodities in the world and it represents the main export from some developing countries. Therefore, the development of new methods of coffee management that improves production without causing any damage to the environment is an attractive alternative for producers. Much effort has been invested towards understanding the mode of action of compounds that can induce resistance against several pathogens without injuring the environment. Many researches have considered silicon efficient in avoiding plant pathogen penetration and development. Our aim was to verify the effect of potassium silicate and calcium/magnesium silicate in the development of coffee seedlings ( Coffea arabica cv. Mundo Novo ) as well as to evaluate the incidence of coffee leaf rust development under greenhouse conditions. The experiment was a completely randomized design with 12 treatments with 10 plants per treatment. The treatments were 0, 0.25, 1.25, 2.5, 4 and 5  μ m of Si for each source of silicon incorporated into the soil. The seedlings were inoculated with a urediniospores suspension of Hemileia vastatrix (2 mg/ml) at the seventh month after planting (six pair of leaves). Evaluations were performed by counting the number of lesions per leaf. The statistical analysis showed that the number of lesions reduced by up to 66% at the highest silicon dose when compared to the number of lesions in control plants. Infected plants were found to have a linear decrease of lesions with the increase of silicate concentration. The lowest number of lesions per leaf area was observed in plants that received 5  μ m of Si from potassium silicate. This result indicates the use of silicon as an alternative for an ecological management system for coffee disease protection.     

Patterns and drivers of fungal disease communities on Arabica coffee along a management gradient

Plants, including those managed by humans, are often attacked by multiple diseases. Yet, most studies focus on single diseases, even if the disease dynamics of multiple species is more interesting from a farmers’ perspective. Moreover, most studies are from single management systems, although it is valuable to understand how diseases are distributed across broad management gradients, especially in cases where less intensive management also provides biodiversity values in the landscape. To understand the spatial dynamics and drivers of diseases across such a broad management gradient, we assessed four major fungal diseases on Arabica coffee (Coffea arabica) at 60 sites in southwestern Ethiopia along a gradient from only little managed wild coffee in the forest understory to intensively managed coffee plantations. We found that environmental and management factors related to disease incidence and severity differed strongly among the four fungal diseases. Coffee leaf rust (Hemileia vastatrix) and Armillaria root rot (Armillaria mellea) were more severe in intensively managed sites, whereas coffee berry disease (Colletotrichum kahawae) and wilt disease (Gibberella xylarioides) were more severe in less managed sites. Among sites, incidence and severity of the four fungal diseases poorly correlated with each other. Within sites, however, shrubs that were severely attacked by coffee leaf rust also had high levels of berry disease symptoms. A better understanding of disease dynamics is important for providing management recommendations that benefit smallholder farmers, but also to evaluate possibilities for maintaining biodiversity values in the landscape related to shade cover complexity and wild coffee genetic variation.     

Genetic diversity in Hemileia vastatrix based on RAPD markers

Random amplified polymorphic DNA (RAPD) was used to assess the genetic structure of Hemileia vastatrix populations. Forty-five rust isolates with different virulence spectra and from different hosts and geographical regions were analyzed. Out of 45 bands, generated with three RAPD primers, 35 (78%) were polymorphic and scored as molecular markers. Cluster analysis exhibits unstructured variability of this pathogen with regard to physiological race, geographical origin or host. The genotypic diversity (H') inferred from Shannon's index was higher than gene diversity (Ht), suggesting that diversity is distributed among clonal lineages. Estimates of gene diversity in Africa and Asia populations were higher in total (Ht) as compared to within population diversity (Hs). Genetic differentiation was considerable among coffee rust isolates from Africa (Gst = 0.865) and Asia (Gst = 0.768) but not among isolates from South America (Gst = 0.266). We concluded that genetic diversity in H. vastatrix was moderately low and that the genetic differentiation among populations shows that asexual reproduction is likely to play an important role in the population biology of this fungus. This should be taken into account for the development of breeding programs.     

Use of fluorescence in situ hybridization as a tool for introgression analysis and chromosome identification in coffee (Coffea arabica L.).

Fluorescence in situ hybridization (FISH) was used to study the presence of alien chromatin in interspecific hybrids and one introgressed line (S.288) derived from crosses between the cultivated species Coffea arabica and the diploid relatives C. canephora and C. liberica. In situ hybridization using genomic DNA from C. canephora and C. arabica as probes showed elevated cross hybridization along the hybrid genome, confirming the weak differentiation between parental genomes. According to our genomic in situ hybridization (GISH) data, the observed genomic resemblance between the modern C. canephora genome (C) and the C. canephora-derived subgenome of C. arabica (Ca) appears rather considerable. Poor discrimination between C and Ca chromosomes supports the idea of low structural modifications of both genomes since the C. arabica speciation, at least in the frequency and distribution of repetitive sequences. GISH was also used to identify alien chromatin segments on chromosome spreads of a C. liberica-introgressed line of C. arabica. Further, use of GISH together with BAC-FISH analysis gave us additional valuable information about the physical localization of the C. liberica fragments carrying the SH3 factor involved in resistance to the coffee leaf rust. Overall, our results illustrate that FISH analysis is a complementary tool for molecular cytogenetic studies in coffee, providing rapid localization of either specific chromosomes or alien chromatin in introgressed genotypes derived from diploid species displaying substantial genomic differentiation from C. arabica.     

Farmers' perceptions, knowledge, and management of coffee pests and diseases and their natural enemies in Chiapas, Mexico

Small farmers' perceptions of coffee Coffea arabica L. herbivores and their natural enemies, how those perceptions relate to field infestation levels, and pest management practices being implemented by members from two organic and nonorganic coffee grower organizations in the Soconusco region, southeastern Mexico, were analyzed through an interview survey, diagnostic workshops, and field sampling. The terms pest, disease, and damage were commonly used as synonyms. The major phytophagous species, as perceived by the interviewees, were Hypothenemus hampei (Ferrari), and to a lesser extent the fungi Corticium koleroga Cooke (H?hnel) and Hemileia vastatrix Berkeley & Broome. Among the nonorganic farmers, other nonpest-related constraints were regarded as more important. Awareness of the existence of natural enemies was low, despite more organic farmers have used the ectoparasitoid bethylid Cephalonomia stephanoderis Betrem against H. hampei. Labor supplied by household members was most frequent for pest control; only organic farmers exchanged labor for this purpose. The levels of infestation by H. hampei, Leucoptera coffeella Guérin-Méneville, and C. koleroga were lower within the organic coffee stands. However, a low effectiveness for pest control was commonly perceived, probably due to a feeling, among the organic farmers, of a low impact of their pest management extension service, whereas a lack of motivation was prevalent among the nonorganic farmers, shown by a concern with their low coffee yields and the emigration of youth. The importance of understanding farmers' perceptions and knowledge of pests and their natural enemies and the need for participatory pest management approaches, are discussed.     

Crude Exopolysaccharides (EPS) from Xanthomonas campestris pv. manihotis, Xanthomonas campestris pv. Campestris and Commercial Xanthan Gum as Inducers of Protection in Coffee Plants against Hemileia vastatrix

Foliar-applied exopolysaccharides, obtained from bacterial cells of either Xanthomonas campestris pv. manihotis (EPS-Xcm) or Xanthomonas campestris pv. campestris (EPS-Xcc), isolate NRRL B-1459, were tested for their ability to induce local and systemic protection against coffee leaf rust caused by Hemileia vastatrix. Both preparations of EPS were effective in inducing local and systemic protection when applied 72 h before challenge with the pathogen. Protection was also observed when plants were treated with different concentrations of a commercially available preparation of xanthan gum (CXG).
Systemic protection was induced by EPS-Xcm, EPS-Xcc and CXG even after the removal of the treated leaves immediately before the challenge. Local protection lasted at least 5 weeks, when EPS-Xcm was applied at the concentration of 100 μg equivalents of glucose/ml. Fluorescent microscopic studies of pathogen development in protected and control leaves indicated that neither the germination, appressoria formation nor the number of infection sites were affected by treatment with EPS-Xcm.     

Chitinases of <Emphasis Type="Italic">Coffea arabica</Emphasis> genotypes resistant to orange rust <Emphasis Type="Italic">Hemileia vastatrix</Emphasis>

Two Coffea arabica — Hemileia vastatrix incompatible interactions (I₁: coffee cv. Caturra — rust race VI and I₂: coffee cv S4 Agaro — rust race II) and a compatible interaction (coffee cv. Caturra — rust race II) were compared in relation to the infection process and chitinase activity. In the two incompatible interactions the fungus ceased growth in the early infection stages, while in the compatible interaction no fungus growth inhibition was observed. A high constitutive level of chitinase activity was detected in the intercellular fluid of healthy leaves. Upon infection, chitinase isoforms were more abundant in incompatible interactions than in the compatible interaction. Immunodetection showed that class I chitinases are particularly relevant in the incompatible interactions and might participate in the defence response of the coffee plants.     

Variation in Aggressiveness Components in the Hemileia vastatrix Population in Brazil

The Pucciniomycete fungus Hemileia vastatrix causes leaf rust on coffee trees. The pathogen is responsible for considerable yield losses in susceptible coffee cultivars if appropriate management strategies are not implemented. Rapid spread and epidemics of rust fungi are usually associated with the emergence of new races of the pathogen that overcome resistance or with the emergence of more aggressive populations of the pathogen. In Brazil, coffee production is dominated by susceptible cultivars of Coffea arabica and Coffea canephora. We assessed aggressiveness in 46 populations of H. vastatrix from Minas Gerais and Espírito Santo, two of the most important coffee‐producing states in Brazil. We observed a significant difference in the incubation period between the populations from Minas Gerais and Espírito Santo when 183 single‐pustule isolates were inoculated onto Catuaí Vermelho IAC 44, a susceptible C. arabica cultivar. Variation in aggressiveness components was observed between and within localities. Isolates with longer incubation periods also tended to have longer latent periods, although there was only a low correlation between these two aggressiveness components (r² = 0.34, P = 2.2 × 10^?16). Low‐sporulating isolates also had significantly longer incubation and latent periods. The H. vastatrix population from Minas Gerais and Espírito Santo is structured by the formation of groups of individuals with differential level of aggressiveness. Our results indicate that the variation in aggressiveness of the Brazilian H. vastatrix population may be associated with the geographic coffee‐producing areas.     

Polyphenoloxidase activity in coffee leaves and its role in resistance against the coffee leaf miner and coffee leaf rust

In plants, PPO has been related to defense mechanism against pathogens and insects and this role was investigated in coffee trees regarding resistance against a leaf miner and coffee leaf rust disease. PPO activity was evaluated in different genotypes and in relation to methyl-jasmonate (Meja) treatment and mechanical damage. Evaluations were also performed using compatible and incompatible interactions of coffee with the fungus Hemileia vastatrix (causal agent of the leaf orange rust disease) and the insect Leucoptera coffeella (coffee leaf miner). The constitutive level of PPO activity observed for the 15 genotypes ranged from 3.8 to 88 units of activity/mg protein. However, no direct relationship was found with resistance of coffee to the fungus or insect. Chlorogenic acid (5-caffeoylquinic acid), the best substrate for coffee leaf PPO, was not related to resistance, suggesting that oxidation of other phenolics by PPO might play a role, as indicated by HPLC profiles. Mechanical damage, Meja treatment, H. vastatrix fungus inoculation and L. coffeella infestation caused different responses in PPO activity. These results suggest that coffee resistance may be related to the oxidative potential of the tissue regarding the phenolic composition rather than simply to a higher PPO activity.     

Ricoseius loxocheles,a phytoseiid mite that feeds on coffee leaf rust

One of the most important diseases of coffee plants is the coffee leaf rust fungus Hemileia vastatrix Berkeley and Broome (Uredinales). It can cause 30 % yield loss in some varieties of Coffea arabica (L.). Besides fungus, the coffee plants are attacked by phytophagous mites. The most common species is the coffee red mite, Oligonychus ilicis McGregor (Acari: Tetranychidae). Predatory mites of the Phytoseiidae family are well-known for their potential to control herbivorous mites and insects, but they can also develop and reproduce on various other food sources, such as plant pathogenic fungi. In a field survey, we found Ricoseius loxocheles (De Leon) (Acari: Phytoseiidae) on the necrotic areas caused by the coffee leaf rust fungus during the reproductive phase of the pathogen. We therefore assessed the development, survivorship and reproduction of R. loxocheles feeding on coffee leaf rust fungus and measured predation and oviposition of this phytoseiid having coffee red mite as prey under laboratory conditions. The mite fed, survived, developed and reproduced successfully on this pathogen but it was not able to prey on O. ilicis. Survival and oviposition with only prey were the same as without food. This phytoseiid mite does not really use O. ilicis as food. It is suggested that R. loxocheles is one phytoseiid that uses fungi as a main food source.     

Origin, diversity and evolution of NBS-type disease-resistance gene homologues in coffee trees (Coffea L.)

The majority of plant disease-resistance genes (R-genes) isolated so far encode a predicted nucleotide-binding site (NBS) domain. NBS domains related to R-genes show a highly conserved backbone of amino acid motifs, which makes it possible to isolate resistance gene analogues (RGAs) by PCR with degenerate primers. Multiple combinations of primers with low degeneracy, designed from two conserved motifs in the NBS regions of R-genes of various plants, were used on genomic DNA from coffee trees, an important perennial tropical crop. Nine distinct classes of RGAs of the NBS-like type, representing a highly diverse sample, were isolated from Coffea arabica and C. canephora species. The analysis of one coffee RGA family suggested point mutations as the primary source of diversity. With one exception, coffee RGA families appeared to be closely related in sequence to at least one cloned R-gene. In addition, deduced amino acid sequences of coffee RGAs were identified that showed strong sequence similarity to almost all known non-TIR (Toll/Interleukin 1 Receptor)-type R-genes. The high degree of similarity between particular coffee RGAs and R-genes isolated from other angiosperm species, such as Arabidopsis, tomato and rice, indicates an ancestral relationship and the existence of common ancestors. The data obtained from coffee species suggests that the evolution of NBS-encoding sequences involves the gradual accumulation of mutations and slow rates of divergence within distinct R-gene families, rather than being a rapid process. Functional inferences drawn from the suggested pattern of evolution of NBS-type R-genes is also discussed.