Metataxonomic Identification of Microorganisms during the Coffee Fermentation Process in Colombian Farms (Cesar Department).

The metataxonomic diversity and microbial composition of microorganisms during the coffee fermentation process as well as their relationship with coffee quality were determined across 20 farms in the department of Cesar, Colombia, by sampling coffee fruits from Coffea arabica; Var. Castillo General®, Var. Colombia, and Var. Cenicafé 1. In each farm, the fruits were processed and the fermentation process took place between 10 and 42 h following this. Three samples of mucilage and washed coffee seeds were collected per farm during the fermentation process. The microorganisms present in the mucilage were identified using metataxonomic methods by amplifying the 16S rRNA gene for bacteria and ITS for fungi. The microorganisms' morphotypes were isolated and identified. The analysis of bacteria allowed for the identification of the following genera: Gluconobacter, Leuconostoc, Acetobacter, Frateuria, Pantoea, Pseudomonas, Tatumella, and Weisella, as well as unclassified enterobacteria; the Lactobacillacea and Secundilactobacillus families were only identified in the Var. Cenicafé 1. For fungi, the top 11 genera and families found included Hanseniaspora, Candida, Meyerozyma, Wickerhamomyces, Pichia, f-Saccharomycodaceae, f-Nectriciae, unclassified fungi, and Saccharomycetaceae, which were only found in Cenicafé 1. A total of 92% of the coffee samples obtained scored between 80.1 and 84.9, indicating "Very Good" coffee (Specialty Coffee Association (SCA) scale). Farms with the longest fermentation times showed better coffee attributes related to acidity, fragrance, and aroma. During coffee fermentation, there is a central microbiome. The differences between the microorganisms' genera could be influenced by the coffee variety, while the specific conditions of each farm (i.e., altitude and temperature) and its fermentation processes could determine the proportions of and interactions between the microbial groups that favor the sensory characteristics responsible for coffee cup quality.

Pilot Testing of an Area-Wide Biological Control Strategy against the Coffee Berry Borer in Colombia Using African Parasitoids.

The coffee berry borer (CBB), Hypothenemus hampei (Ferrari, 1867) (Coleoptera: Curculionidae: Scolytinae), native to Africa, is a major global insect pest of coffee. It has invaded many coffee production areas around the world that do not have natural enemies. In this study, two African parasitoids, Prorops nasuta Waterston (Hymenoptera: Bethylidae) and Phymastichus coffea Waterston (Hymenoptera: Eulophidae), were mass-reared for field release against H. hampei in Chinchiná, Colombia. More than 1.5 million wasps of each species were released on a 61-hectare coffee farm in replicated plots, resulting in parasitism rates of up to 7.7% for P. nasuta and 56.3% for P. coffea. This led to a maximum reduction in H. hampei field populations of 81% from dispersal coffee plots (old coffee crops before plant stumping) and 64.3% in colonization coffee plots (new coffee crops with active growing and fruiting plants) within the farm. As a result of this area-wide strategy, the percentage of CBB-infested coffee berries in colonization coffee plots decreased from 51.1 to 77.5% compared to coffee plots without parasitoid releases. This approach offers a promising alternative to the use of chemical insecticides and could be integrated into current pest management programs to control H. hampei.

Structure and Dynamics of the Gut Bacterial Community Across the Developmental Stages of the Coffee Berry Borer, Hypothenemus hampei.

The coffee berry borer (CBB); Hypothenemus hampei (Coleoptera: Curculionidae), is widely recognized as the major insect pest of coffee crops. Like many other arthropods, CBB harbors numerous bacteria species that may have important physiological roles in host nutrition, detoxification, immunity and protection. To date, the structure and dynamics of the gut-associated bacterial community across the CBB life cycle is not yet well understood. A better understanding of the complex relationship between CBB and its bacterial companions may provide new opportunities for insect control. In the current investigation, we analyzed the diversity and abundance of gut microbiota across the CBB developmental stages under field conditions by using high-throughput Illumina sequencing of the 16S ribosomal RNA gene. Overall, 15 bacterial phyla, 38 classes, 61 orders, 101 families and 177 genera were identified across all life stages, including egg, larva 1, larva 2, pupa, and adults (female and male). Proteobacteria and Firmicutes phyla dominated the microbiota along the entire insect life cycle. Among the 177 genera, the 10 most abundant were members of Ochrobactrum (15.1%), Pantoea (6.6%), Erwinia (5.7%), Lactobacillus (4.3%), Acinetobacter (3.4%), Stenotrophomonas (3.1%), Akkermansia (3.0%), Agrobacterium (2.9%), Curtobacterium (2.7%), and Clostridium (2.7%). We found that the overall bacterial composition is diverse, variable within each life stage and appears to vary across development. About 20% of the identified OTUs were shared across all life stages, from which 28 OTUs were consistently found in all life stage replicates. Among these OTUs there are members of genera Pantoea, Erwinia, Agrobacterium, Ochrobactrum, Pseudomonas, Acinetobacter, Brachybacterium, Sphingomonas and Methylobacterium, which can be considered as the gut-associated core microbiota of H. hampei. Our findings bring additional data to enrich the understanding of gut microbiota in CBB and its possible use for development of insect control strategies.

A coffee berry borer (Hypothenemus hampei) genome assembly reveals a reduced chemosensory receptor gene repertoire and male-specific genome sequences.

Coffee berry borer-CBB (Hypothenemus hampei) is a globally important economic pest of coffee (Coffea spp.). Despite current insect control methods for managing CBB, development of future control strategies requires a better understanding of its biology and interaction with its host plant. Towards this objective, we performed de novo CBB genome and transcriptome sequencing, improved CBB genome assembly and predicted 18,765 protein-encoding genes. Using genome and transcriptome data, we annotated the genes associated with chemosensation and found a reduced gene repertoire composed by 67 odorant receptors (ORs), 62 gustatory receptors (GRs), 33 ionotropic receptors (IRs) and 29 odorant-binding proteins (OBPs). In silico transcript abundance analysis of these chemosensory genes revealed expression enrichment in CBB adults compared with larva. Detection of differentially expressed chemosensory genes between males and females is likely associated with differences in host-finding behavior between sexes. Additionally, we discovered male-specific genome content and identified candidate male-specific expressed genes on these scaffolds, suggesting that a Y-like chromosome may be involved in the CBB's functional haplodiploid mechanism of sex determination.

BSA-Seq Discovery and Functional Analysis of Candidate Hessian Fly (Mayetiola destructor) Avirulence Genes.

The Hessian fly (HF, Mayetiola destructor) is a plant-galling parasite of wheat (Triticum spp.). Seven percent of its genome is composed of highly diversified signal-peptide-encoding genes that are transcribed in HF larval salivary glands. These observations suggest that they encode effector proteins that are injected into wheat cells to suppress basal wheat immunity and redirect wheat development towards gall formation. Genetic mapping has determined that mutations in four of these genes are associated with HF larval survival (virulence) on plants carrying four different resistance (R) genes. Here, this line of investigation was pursued further using bulked-segregant analysis combined with whole genome resequencing (BSA-seq). Virulence to wheat R genes H6, Hdic, and H5 was examined. Mutations associated with H6 virulence had been mapped previously. Therefore, we used H6 to test the capacity of BSA-seq to map virulence using a field-derived HF population. This was the first time a non-structured HF population had been used to map HF virulence. Hdic virulence had not been mapped previously. Using a structured laboratory population, BSA-seq associated Hdic virulence with mutations in two candidate effector-encoding genes. Using a laboratory population, H5 virulence was previously positioned in a region spanning the centromere of HF autosome 2. BSA-seq resolved H5 virulence to a 1.3 Mb fragment on the same chromosome but failed to identify candidate mutations. Map-based candidate effectors were then delivered to Nicotiana plant cells via the type III secretion system of Burkholderia glumae bacteria. These experiments demonstrated that the genes associated with virulence to wheat R genes H6 and H13 are capable of suppressing plant immunity. Results are consistent with the hypothesis that effector proteins underlie the ability of HFs to survive on wheat.

Genome-wide analysis of transposable elements in the coffee berry borer Hypothenemus hampei (Coleoptera: Curculionidae): description of novel families.

The coffee berry borer (CBB) Hypothenemus hampei is the most limiting pest of coffee production worldwide. The CBB genome has been recently sequenced; however, information regarding the presence and characteristics of transposable elements (TEs) was not provided. Using systematic searching strategies based on both de novo and homology-based approaches, we present a library of TEs from the draft genome of CBB sequenced by the Colombian Coffee Growers Federation. The library consists of 880 sequences classified as 66% Class I (LTRs: 46%, non-LTRs: 20%) and 34% Class II (DNA transposons: 8%, Helitrons: 16% and MITEs: 10%) elements, including families of the three main LTR (Gypsy, Bel-Pao and Copia) and non-LTR (CR1, Daphne, I/Nimb, Jockey, Kiri, R1, R2 and R4) clades and DNA superfamilies (Tc1-mariner, hAT, Merlin, P, PIF-Harbinger, PiggyBac and Helitron). We propose the existence of novel families: Hypo, belonging to the LTR Gypsy superfamily; Hamp, belonging to non-LTRs; and rosa, belonging to Class II or DNA transposons. Although the rosa clade has been previously described, it was considered to be a basal subfamily of the mariner family. Based on our phylogenetic analysis, including Tc1, mariner, pogo, rosa and Lsra elements from other insects, we propose that rosa and Lsra elements are subfamilies of an independent family of Class II elements termed rosa. The annotations obtained indicate that a low percentage of the assembled CBB genome (approximately 8.2%) consists of TEs. Although these TEs display high diversity, most sequences are degenerate, with few full-length copies of LTR and DNA transposons and several complete and putatively active copies of non-LTR elements. MITEs constitute approximately 50% of the total TEs content, with a high proportion associated with DNA transposons in the Tc1-mariner superfamily.

Avirulence gene mapping in the Hessian fly (Mayetiola destructor) reveals a protein phosphatase 2C effector gene family.

The genetic tractability of the Hessian fly (HF, Mayetiola destructor) provides an opportunity to investigate the mechanisms insects use to induce plant gall formation. Here we demonstrate that capacity using the newly sequenced HF genome by identifying the gene (vH24) that elicits effector-triggered immunity in wheat (Triticum spp.) seedlings carrying HF resistance gene H24. vH24 was mapped within a 230-kb genomic fragment near the telomere of HF chromosome X1. That fragment contains only 21 putative genes. The best candidate vH24 gene in this region encodes a protein containing a secretion signal and a type-2 serine/threonine protein phosphatase (PP2C) domain. This gene has an H24-virulence associated insertion in its promoter that appears to silence transcription of the gene in H24-virulent larvae. Candidate vH24 is a member of a small family of genes that encode secretion signals and PP2C domains. It belongs to the fraction of genes in the HF genome previously predicted to encode effector proteins. Because PP2C proteins are not normally secreted, our results suggest that these are PP2C effectors that HF larvae inject into wheat cells to redirect, or interfere, with wheat signal transduction pathways.