Annotation of a hybrid partial genome of the coffee rust (Hemileia vastatrix) contributes to the gene repertoire catalog of the Pucciniales.

Coffee leaf rust caused by the fungus Hemileia vastatrix is the most damaging disease to coffee worldwide. The pathogen has recently appeared in multiple outbreaks in coffee producing countries resulting in significant yield losses and increases in costs related to its control. New races/isolates are constantly emerging as evidenced by the presence of the fungus in plants that were previously resistant. Genomic studies are opening new avenues for the study of the evolution of pathogens, the detailed description of plant-pathogen interactions and the development of molecular techniques for the identification of individual isolates. For this purpose we sequenced 8 different H. vastatrix isolates using NGS technologies and gathered partial genome assemblies due to the large repetitive content in the coffee rust hybrid genome; 74.4% of the assembled contigs harbor repetitive sequences. A hybrid assembly of 333 Mb was built based on the 8 isolates; this assembly was used for subsequent analyses. Analysis of the conserved gene space showed that the hybrid H. vastatrix genome, though highly fragmented, had a satisfactory level of completion with 91.94% of core protein-coding orthologous genes present. RNA-Seq from urediniospores was used to guide the de novo annotation of the H. vastatrix gene complement. In total, 14,445 genes organized in 3921 families were uncovered; a considerable proportion of the predicted proteins (73.8%) were homologous to other Pucciniales species genomes. Several gene families related to the fungal lifestyle were identified, particularly 483 predicted secreted proteins that represent candidate effector genes and will provide interesting hints to decipher virulence in the coffee rust fungus. The genome sequence of Hva will serve as a template to understand the molecular mechanisms used by this fungus to attack the coffee plant, to study the diversity of this species and for the development of molecular markers to distinguish races/isolates.

In vitro production of two chitinolytic proteins with an inhibiting effect on the insect coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae) and the fungus Hemileia vastatrix the most limiting pests of coffee crops.

Two genes from Streptomyces albidoflavus, one exochitinase (905-bp) and an endochitinase (1100-bp) were functionally expressed in Escherichia coli in form of a fusion protein with a maltose binding protein (MBP). The goal was to produce and test proteins that inhibit both the coffee berry borer insect Hypothenemus hampei and the coffee rust fungus Hemileia vastatrix. Both recombinant proteins MBP/exochitinase and MBP/endochitinase showed chitinolytic activity. When recombinant purified proteins were added to an artificial coffee-based diet for the coffee berry borer, MBP/exochitinase at a concentration of 0.5% W/W caused delayed growth of larvae and 100% mortality between days 8 and 15, while MBP/endochitinase caused 100% mortality at day 35. H. vastatrix urediniospores presented total cell wall degradation in their germinative tubes within 18 h of exposure to the proteins at enzyme concentrations of 5 and 6 mg ml-1, with exochitinase having the greatest effect. The dual deleterious effect of S. albidoflavus chitinases on two of the most limiting coffee pests worldwide, the coffee borer and the coffee rust, make them potential elements to be incorporated in integrated control strategies.

Transcriptome analysis of the entomopathogenic fungus Beauveria bassiana grown on cuticular extracts of the coffee berry borer (Hypothenemus hampei).

The coffee berry borer (CBB; Hypothenemus hampei) is a major pest of coffee responsible for significant crop losses worldwide. The entomopathogenic fungus Beauveria bassiana represents a natural means of controlling this insect pest; however, little is known concerning the molecular determinants that contribute to the virulence of the fungus towards the CBB. In order to examine genes involved in insect virulence, two expressed sequence tag (EST) libraries, representing germinating conidia and growing hyphae/mycelia of B. bassiana cells grown on cuticular extracts of the CBB were constructed and analysed. In total, 4186 cDNA transcripts were obtained, which included 2141 from the cuticle-germinated conidia and 2045 from the cuticle-grown mycelium libraries, respectively. The average sequence length obtained was 470 bp and transcript assembly resulted in a set of 1271 and 1305 unique gene sequences for the conidial and mycelia libraries, respectively. Around 50 % of the sequences in each library could be annotated by gene ontology terms. An analysis of the two generated libraries as well as a previously reported EST library of B. bassiana grown on chitin was performed. Between the cuticle-germinated conidia and the cuticle-grown mycelia libraries, 322 unique gene sequences were shared, of which 90 % could be annotated, leaving 949 unique cuticle-germinated conidial genes and 983 unique growing hyphae/mycelia genes of which around 65 % were annotated. ESTs shared between the libraries indicated a basic response pattern for B. bassiana against H. hampei, which included genes implicated in pathogenicity. The expression profiles of four genes were evaluated with a cyclophilin, an alkaline-like serine protease and a mitogen-activated protein kinase (MAPK), showing elevated expression during initial phases of infection, i.e. conidia germinating on insect extracts. These data provide clues and gene candidates for further exploration concerning the biology and molecular mechanisms of entomopathogenicity by this fungus.

Detection and Identification of a Group 16SrIII-Related Phytoplasma Associated with Coffee Crispiness Disease in Colombia.

Coffee crispiness ("crespera"), a disease of uncertain etiology, has been endemic in coffee (Coffea arabica L.) plantations in Colombia for at least 60 years. Symptoms typically consist of bud proliferation, abundant short and narrow leaves, phyllody, floral abortion, monospermic fruit, and dwarfing of plants. In severe cases, coffee crispiness disease (CCD) can affect production significantly. In this study, association of a phytoplasma with CCD was indicated by the accumulation of Diene's stain, or 4', 6-diamidino-2-phenylindole fluorescence, only in phloem of affected plant tissues. The presence of polymorphic phytoplasma cells in phloem sieve tube elements was confirmed by transmission electron microscopy. The disease was transmitted successfully by grafting symptomatic shoots from CCD-affected C. arabica plants onto young, healthy rootstocks; however, symptoms failed to develop after mechanical inoculation of young plants with extracts derived from diseased plant tissues. A nested polymerase chain reaction (PCR) assay employing primer pairs P1/P7 followed by FU5/rU3 amplified a 16S ribosomal DNA product (941 bp) exclusively from DNA of diseased plants. Sequence and phylogenetic analysis of the nested PCR product identified the CCD phytoplasma as a new strain member of group 16SrIII (X-disease group). This is the first report of a phytoplasma infecting coffee plants.

Exploiting the genetic diversity of Beauveria bassiana for improving the biological control of the coffee berry borer through the use of strain mixtures.

Beauveria bassiana is an entomopathogen widely used to control the coffee berry borer in Colombia, as part of an Integrated Pest Management strategy. Traditionally, the development of fungal insect pathogens as biocontrol agents in crop pests has been oriented towards the selection and formulation of elite clonal strains. Instead, we explored the potential application of genetic diversity in B. bassiana by determining the effect of strain mixtures on coffee berry borer mortality compared to clonal isolates. Genomic DNA from 11 strains was characterized using internal transcribed spacers and beta-tubulin sequences as well as amplified fragment length polymorphism markers. Cluster analysis produced three genetic groups and confirmed the low but significant intraspecific genetic diversity present among the strains. Single strain virulence towards the coffee berry borer under laboratory conditions, using 1x10(6) conidia ml(-1), ranged between 89.9 and 57.5%. All the inoculations with mixtures resulted in coinfection events. Combinations of genetically similar strains showed no significant differences when their virulences were compared. However, mixtures of genetically different strains led to both antagonism and synergism. The lowest virulence percentage (57%) was obtained by putting together the most virulent strain of each group, contrary to the highest virulence percentage (93%) that resulted from mixing the three least virulent strains. The results indicate the promising potential of designing strain mixtures as an alternative for the biocontrol of Hypothenemus hampei and other pests and provide tools for the understanding of the ecological dynamics of entomopathogen populations under natural conditions.