Coffee Fruit Rot: The Previously Unrecognized Role of Fusarium and Its Interactions with the Coffee Berry Borer (Hypothenemus hampei).

Coffee fruit rot (CFR) is a well-known disease worldwide, mainly caused by Colletotrichum spp., the most important species being C. kahawae subsp. kahawae. In Puerto Rico, Colletotrichum spp. were identified as pathogens of coffee fruits. The coffee berry borer (CBB) was shown to be a dispersal agent of these fungi, and interaction of Fusarium with Colletotrichum affecting coffee fruits was suggested. In this study, we demonstrated that Fusarium spp. also cause CFR in Puerto Rico. Fusarium spp. are part of the CBB mycobiota, and this insect is responsible for spreading the pathogens in coffee fields. We identified nine Fusarium spp. (F. nirenbergiae, F. bostrycoides, F. crassum, F. hengyangense, F. solani-melongenae, F. pseudocircinatum, F. meridionale, F. concolor, and F. lateritium) belonging to six Fusarium species complexes isolated from CBBs and from rotten coffee fruits. Pathogenicity tests showed that F. bostrycoides, F. lateritium, F. nirenbergiae, F. solani-melongenae, and F. pseudocircinatum were pathogens causing CFR on green coffee fruits. F. bostrycoides was the predominant species isolated from the CBB mycobiota and coffee fruits with symptoms of CFR, suggesting a close relationship between F. bostrycoides and the CBB. To our knowledge, this is the first report of F. bostrycoides, F. solani-melongenae, F. pseudocircinatum, and F. nirenbergiae causing CFR worldwide and the first report of F. lateritium causing CFR in Puerto Rico. Understanding the CFR disease complex and how the CBB contributes to dispersing different Fusarium spp. on coffee farms is important to implement disease management practices in Puerto Rico and in other coffee-producing countries.

Morphological and Physio-Chemical Responses to PEG-Induced Water Stress in Vanilla planifolia and V. pompona Hybrids.

Vanilla planifolia is an orchid of cultural and economic value. However, its cultivation in many tropical countries is threatened by water stress. In contrast, V. pompona is a species that is tolerant of prolonged periods of drought. Due to the need for plants' resistant to water stress, the use of hybrids of these two species is considered. Therefore, the objective of this study was to evaluate the morphological and physio-chemical responses of in vitro vanilla seedlings of the parental genotype V. planifolia, and the hybrids V. planifolia × V. pompona and V. pompona × V. planifolia, which were then exposed over five weeks to polyethylene glycol-induced water stress (-0.49 mPa). Stem and root length, relative growth rate, number of leaves and roots, stomatal conductance, specific leaf area, and leaf water content were determined. Metabolites potentially associated with the response to water stress were identified in leaves, through untargeted and targeted metabolomics. Both hybrids exhibited a smaller decrease in the morphophysiological responses compared to V. planifolia and exhibited an enrichment of metabolites such as carbohydrates, amino acids, purines, phenols, and organic acids. Hybrids of these two species are considered as a potential alternative to the traditional cultivation of vanilla to face drought in a global warming scenario.

Pathogens Causing Anthracnose and Fruit Rots of Coffee Associated with the Coffee Berry Borer and the Entomopathogenic Fungus Beauveria bassiana in Puerto Rico.

Fruit rots reduce coffee production worldwide. Eight Colletotrichum species have been reported to cause coffee fruit rots; the most important is C. kahawae, the cause of coffee berry disease (CBD) in Africa. It is unknown whether these fruit rot pathogens can be dispersed by the coffee berry borer (CBB, Hypothenemus hampei) or whether Beauveria bassiana (a natural enemy of CBB) might reduce coffee fruit rots. We identified pathogens causing coffee fruits rots in Puerto Rico and evaluated whether B. bassiana reduced fruit rot and whether CBB could disperse pathogens. A total of 2,333 coffee fruit with CBB damage were collected; of these, 1,197 had visible growth of B. bassiana. C. fructicola, C. siamense, C. theobromicola, and C. tropicale were isolated and identified from the fruit using morphological traits and phylogeny of three nuclear genes. All four species caused internal and external rot after inoculation of healthy green coffee fruit. Coffee fruit treated with B. bassiana had significantly less fruit rot than untreated fruit, suggesting B. bassiana can protect against fruit rot. To test whether B. bassiana had a protective effect, B. bassiana and Colletotrichum were coinoculated on coffee fruit. Fruit inoculated with both B. bassiana and Colletotrichum had significantly less rot than fruit inoculated with Colletotrichum alone. To test if CBBs dispersed the pathogens, CBBs were exposed to Colletotrichum conidia and placed on green fruit, which resulted in fruit rot. This study identifies new pathogens causing coffee fruit rot, shows that C. kahawae is not the only Colletotrichum that attacks green fruits, suggests a role for B. bassiana in disease management and demonstrates CBB can disperse the pathogens.

The Coffee Berry Borer (Coleoptera: Curculionidae) in Puerto Rico: Distribution, Infestation, and Population per Fruit.

The coffee berry borer (CBB) (Hypothenemus hampei: Ferrar) was first detected in Puerto Rico in 2007. Its distribution since then has been extensive, but not extensively documented. An island-wide survey was carried out from August to November 2014 (the coffee production season) to assess CBB distribution, infestation, and population per fruit. The CBB was well-established throughout the coffee-growing area of Puerto Rico, but was not evenly distributed. Infestation (or percentages of fruits perforated) in sites sampled ranged from 0 to 95%, and CBB number per infested fruit varied from 1 to 34 individuals. CBB infestation and total population per fruit were positively correlated with altitude. Highest infestation and total population were observed in sites located >400 masl; most of the coffee-producing area in Puerto Rico is above this altitude. Coffea arabica (L.) had higher CBB infestation and population per fruit than Coffea canephora (Pierre ex A. Froehner) (robusta coffee). Based on these results, management tools should be implemented to mitigate the severe damage that CBB is causing in Puerto Rico. These management tools should include the removal of all fruits that remain on the plants after harvest and the use of the entomopathogenic fungus Beauveria bassiana (Balls.) Vuill. for biocontrol, especially on coffee farms at higher elevations.

Age-dependent mycorrhizal specificity in an invasive orchid, Oeceoclades maculata.

PREMISE OF THE STUDY: Oeceoclades maculata is a naturalized, invasive, terrestrial orchid in Puerto Rico and elsewhere in the neotropics. We asked whether its success might be partly explained by its mycorrhizal associations, hypothesizing a relationship with many fungal partners or with one widely distributed partner. METHODS: Oeceoclades maculata roots were collected throughout Puerto Rico, and the degree of mycorrhizal colonization was measured. For identification of fungi, the ITS region was sequenced from pure cultures and directly from roots. Representative fungi were used for symbiotic seed germination experiments. KEY RESULTS: Colonization of O. maculata roots was very variable. The most common fungus identified by BLAST searches was Psathyrella cf. candolleana, but typical orchid mycorrhizal fungi (Ceratobasidium and Tulasnella) were also found, as were a range of saprotrophs. Seeds germinated in vitro only in the presence of Psathyrella. CONCLUSIONS: These results are surprising in two respects. First, O. maculata appears to be highly specific for fungi during seed germination, but unusually promiscuous as adult plants. Second, mycorrhizal associations with Psathyrella and with other saprotrophic fungi have been previously reported, but only from mycoheterotrophic (i.e., nonphotosynthetic) orchids, not from green orchids like Oeceoclades. This combination may partly explain the success of Oeceoclades.

An opportunistic human pathogen on the fly: strains of Aspergillus flavus vary in virulence in Drosophila melanogaster.

Aspergilloses are fungal diseases in humans and animals that is caused by members of the genus Aspergillus. Aspergillus flavus is an important opportunistic pathogen, second only to A. fumigatus as a cause of human aspergillosis. Differences in virulence among A. flavus isolates from clinical and other substrates and mating types are not well known. The fruit fly Drosophila melanogaster has become a model organism for investigating virulence of human pathogens due to similarities between its immune system and that of mammals. In this study we used D. melanogaster as a model host to compare virulence among A. flavus strains obtained from clinical sources as compared with other substrates, between isolates of different mating types, and between isolates of A. flavus and A. fumigatus. Anesthetized flies were infected with A. flavus; mortality ranged from 15% to >90%. All strains were virulent, but some were significantly more so than others, which in turn led to the wide mortality range. Clinical strains were significantly less virulent than environmental strains, probably because the clinical strains were from culture collections and the environmental strains were recent isolates. Mean virulence did not differ between MAT1-1 and MAT1-2 mating types and the phylogeny of A. flavus isolates did not predict virulence. A. flavus was on average significantly more virulent than A. fumigatus on two lines of wild-type flies, Canton-S and Oregon-R. D. melanogaster is an attractive model to test pathogenicity and could be useful for identifying genes involved in virulence.

The double life of Ceratobasidium: orchid mycorrhizal fungi and their potential for biocontrol of Rhizoctonia solani sheath blight of rice.

Ceratobasidium includes orchid mycorrhizal symbionts, plant pathogens and biocontrol agents of soilborne plant pathogens. It is not known to what extent members of the first guild also can participate in the others. Ceratobasidium spp. were isolated from roots of Colombian orchids and identified by phylogeny based on nrITS sequences. Phylogenetic grouping of Ceratobasidium spp. isolates corresponded to orchid host substrate (epiphytic vs. terrestrial). Isolates were tested for virulence on rice and for biocontrol of Rhizoctonia solani, causal agent of sheath blight of rice. All Ceratobasidium spp. isolates caused some signs of sheath blight but significantly less than a pathogenic R. solani used as a positive control. When Ceratobasidium spp. isolates were inoculated on rice seedlings 3 d before R. solani, they significantly reduced disease expression compared to controls inoculated with R. solani alone. The use of Ceratobasidium spp. from orchids for biological control is novel, and biodiverse countries such as Colombia are promising places to look for new biocontrol agents.

Establishing an Integrated Pest Management Program for Coffee Berry Borer (Hypothenemus hampei) in Hawaii and Puerto Rico Coffee Agroecosystems: Achievements and Challenges.

Coffee berry borer (CBB) is the most serious insect pest of coffee worldwide, causing significant reductions in yield and quality. Following the introduction of CBB to Puerto Rico (2007) and Hawaii (2010), researchers, extension agents, industry representatives, and coffee growers have worked together to develop an integrated pest management (IPM) program that is both effective and economically feasible for each island. Since the introduction of the IPM program in Hawaii, research efforts have led to a better understanding of CBB population dynamics, as well as optimized monitoring, cultural practices, and commercial Beauveria bassiana applications. As a result of these efforts, a substantial reduction in average CBB infestation and an increase in coffee yields and quality have been documented in Hawaii over the last decade. However, significant challenges remain in addressing high production and labor costs, limited availability of labor, and a lack of training for field workers in both regions. Although considerable effort has gone into research to support CBB IPM in Hawaii and Puerto Rico, the adoption of these strategies by coffee farmers needs to be increased. More diversified methods of outreach and education are needed to reach growers in rural, isolated areas. Significant gaps exist in the ability and willingness of growers and workers to access and digest information online, emphasizing the importance of on-farm workshops and farmer-to-farmer teaching. Additional methods of training are needed to help coffee farmers and field workers learn how to properly conduct cultural controls and optimize the use of biological control agents such as B. bassiana.

Demography and perturbation analyses of the coffee berry borer Hypothenemus hampei (Coleoptera: Curculionidae): Implications for management.

The coffee berry borer (CBB) Hypothenemus hampei Ferrari is the most serious pest of coffee worldwide. Management of the CBB is extremely difficult because its entire life cycle occurs inside the fruit, where it is well protected. Knowing which life stages contribute most to population growth, would shed light on the population dynamics of this pest and help to improve CBB management programs. Two staged-classified matrices were constructed for CBB populations reared in the lab on artificial diets and CBB populations from artificial infestations in the field. Matrices were used to determine demographic parameters, to conduct elasticity analyses, and to perform prospective perturbation analysis. Higher values of the intrinsic rate of natural increase (rm) and population growth rate (λ): were observed for CBB populations growing in the lab than in the field (rm: 0.058, λ: 1.74 lab; rm: 0.053, λ: 1.32 field). Sensitivity values for both CBB populations were highest for the transitions from larva to pupa (G2: 0.316 lab, 0.352 field), transition from pupa to juvenile (G3: 0.345 lab, 0.515 field) and survival of adult females (P5: 0.324 lab, 0.389 field); these three vital rates can be important targets for CBB management. Prospective perturbation analyses indicated that an effective management for the CBB should consider multiple developmental stages; perturbations of >90% for each transition are necessary to reduce λ to <1. However, when the three vital rates with highest sensitivity are impacted at the same time, the percentage of perturbation is reduced to 25% for each transition; with these reductions in survival of larvae, pupae and adult females the value of λ was reduced from 1.32 to 0.96. Management programs for CBB should be focused on the use of biological and cultural measures that are known to affect these three important targets.

A one-dimensional map to study multi-seasonal coffee infestation by the coffee berry borer.

The coffee berry borer (CBB, Hypothenemus hampei) is the most serious insect pest of coffee worldwide; understanding the dynamics of its reproduction is essential for pest management. The female CBB penetrates the coffee berry, eats the seed, and reproduces inside it. A mathematical model of the infestation progress of the coffee berry by the CBB during several coffee seasons is formulated. The model represents the interaction among five populations: uninfested, slightly infested, and severely infested coffee berries, and free and encapsulated CBBs. Coffee harvesting is also included in the model. A one-dimensional map is derived for tracking the population dynamics subject to certain coffee harvesting percentages over several seasons. Stability analysis of the map's fixed points shows that CBB infestation could be eliminated or controlled to a specific level over multiple seasons of coffee harvesting. However, the percent of coffee harvesting required is determined by the level of CBB infestation at the beginning of the first season and in some cases it is impossible to achieve that percentage.

Gene expression on the fly: A transcriptome-level view of Drosophila's immune response to the opportunistic fungal pathogen Aspergillus flavus.

Aspergilloses are opportunistic infections in animals and humans caused by several Aspergillus species, including Aspergillus flavus. Although the immune system of Drosophila melanogaster is extensively studied, little is known about the fly's specific responses to infection by A. flavus. We compared gene expression levels during induced infections in D. melanogaster by a virulent A. flavus isolate and a less virulent isolate, as well as from uninfected flies as a control. We found that 1081 of the 14,554 gene regions detected were significantly differentially expressed among treatments. Some of these up- and down- regulated genes were previously shown to be involved in defense responses against pathogens. Some are known to be involved in vitelline membrane formation in flies. Other up- and down-regulated genes are of unknown function. Understanding expression of these genes during the process of infection in flies should improve our knowledge of innate immunity in invertebrates, and by extension, in vertebrates as well.

Mycorrhizal fungi of Vanilla: diversity, specificity and effects on seed germination and plant growth.

Mycorrhizal fungi are essential for the germination of orchid seeds. However, the specificity of orchids for their mycorrhizal fungi and the effects of the fungi on orchid growth are controversial. Mycorrhizal fungi have been studied in some temperate and tropical, epiphytic orchids, but the symbionts of tropical, terrestrial orchids are still unknown. Here we study diversity, specificity and function of mycorrhizal fungi in Vanilla, a pantropical genus that is both terrestrial and epiphytic. Mycorrhizal roots were collected from four Vanilla species in Puerto Rico, Costa Rica and Cuba. Cultured and uncultured mycorrhizal fungi were identified by sequencing the internal transcribed spacer region of nuclear rDNA (nrITS) and part of the mitochondrial ribosomal large subunit (mtLSU), and by counting number of nuclei in hyphae. Vanilla spp. were associated with a wide range of mycorrhizal fungi: Ceratobasidium, Thanatephorus and Tulasnella. Related fungi were found in different species of Vanilla, although at different relative frequencies. Ceratobasidium was more common in roots in soil and Tulasnella was more common in roots on tree bark, but several clades of fungi included strains from both substrates. Relative frequencies of genera of mycorrhizal fungi differed significantly between cultured fungi and those detected by direct amplification. Ceratobasidium and Tulasnella were tested for effects on seed germination of Vanilla and effects on growth of Vanilla and Dendrobium plants. We found significant differences among fungi in effects on seed germination and plant growth. Effects of mycorrhizal fungi on Vanilla and Dendrobium were similar: a clade of Ceratobasidium had a consistently positive effect on plant growth and seed germination. This clade has potential use in germination and propagation of orchids. Results confirmed that a single orchid species can be associated with several mycorrhizal fungi with different functional consequences for the plant.

Wolbachia Affects Reproduction and Population Dynamics of the Coffee Berry Borer (Hypothenemus hampei): Implications for Biological Control.

Wolbachia are widely distributed endosymbiotic bacteria that influence the reproduction and fitness of their hosts. In recent years the manipulation of Wolbachia infection has been considered as a potential tool for biological control. The coffee berry borer (CBB), Hypothenemus hampei, is the most devastating coffee pest worldwide. Wolbachia infection in the CBB has been reported, but until now the role of Wolbachia in CBB reproduction and fitness has not been tested. To address this issue we reared the CBB in artificial diets with and without tetracycline (0.1% w/v) for ten generations. Tetracycline reduced significantly the relative proportion of Wolbachia in the CBB microbiota from 0.49% to 0.04%. This reduction affected CBB reproduction: females fed with tetracycline had significantly fewer progeny, lower fecundity, and fewer eggs per female. Tetracycline also reduced the population growth rate (λ), net reproductive rate (R0), and mean generation time (T) in CBB; the reduction in population growth was mostly due to variation in fertility, according to life time response experiments (LTREs) analysis. Our results suggest that Wolbachia contribute to the reproductive success of the CBB and their manipulation represents a possible approach to CBB biocontrol mediated by microbiome management.

Differential Microbial Diversity in Drosophila melanogaster: Are Fruit Flies Potential Vectors of Opportunistic Pathogens?

Drosophila melanogaster has become a model system to study interactions between innate immunity and microbial pathogens, yet many aspects regarding its microbial community and interactions with pathogens remain unclear. In this study wild D. melanogaster were collected from tropical fruits in Puerto Rico to test how the microbiota is distributed and to compare the culturable diversity of fungi and bacteria. Additionally, we investigated whether flies are potential vectors of human and plant pathogens. Eighteen species of fungi and twelve species of bacteria were isolated from wild flies. The most abundant microorganisms identified were the yeast Candida inconspicua and the bacterium Klebsiella sp. The yeast Issatchenkia hanoiensis was significantly more common internally than externally in flies. Species richness was higher in fungi than in bacteria, but diversity was lower in fungi than in bacteria. The microbial composition of flies was similar internally and externally. We identified a variety of opportunistic human and plant pathogens in flies such as Alcaligenes faecalis, Aspergillus flavus, A. fumigatus, A. niger, Fusarium equiseti/oxysporum, Geotrichum candidum, Klebsiella oxytoca, Microbacterium oxydans, and Stenotrophomonas maltophilia. Despite its utility as a model system, D. melanogaster can be a vector of microorganisms that represent a potential risk to plant and public health.

Integrated Pest Management of Coffee Berry Borer in Hawaii and Puerto Rico: Current Status and Prospects.

The coffee berry borer (CBB), Hypothenemus hampei, is the most significant insect pest of coffee worldwide. Since CBB was detected in Puerto Rico in 2007 and Hawaii in 2010, coffee growers from these islands are facing increased costs, reduced coffee quality, and increased pest management challenges. Here, we outline the CBB situation, and summarize the findings of growers, researchers, and extension professionals working with CBB in Hawaii. Recommendations for the Integrated Pest Management (IPM) program for CBB in Hawaiian Islands and Puerto Rico include: (1) establish a CBB monitoring program, (2) synchronize applications of insecticides with peak flight activity of CBB especially during the early coffee season, (3) conduct efficient strip-picking as soon as possible after harvest and perform pre-harvest sanitation picks in CBB hotspots if needed, (4) establish protocols to prevent the escape of CBB from processing areas and when transporting berries during harvest, and (5) stump prune by blocks. Progress achieved includes the introduction of the mycoinsecticide Beauveria bassiana to coffee plantations, the coordination of area-wide CBB surveys, the establishment and augmentation of native beetle predators, and an observed reduction of CBB populations and increased coffee quality where IPM programs were established. However, CBB remains a challenge for coffee growers due to regional variability in CBB pressures, high costs, and labor issues, including a lack of training and awareness of CBB management practices among growers.

Forensic Phytopathology: a Critical Review.

Forensic phytopathology is the application of plant pathology to legal or criminal matters. It is an emerging field. The existing literature focuses mainly on potential agricultural bioterrorism threats to the United States. Here we try to take a broader view including agricultural bioterrorism, mycoherbicide applications to eradicate plants used for illegal drugs, civil cases involving charges of sale or movement of diseased plants, and mycotoxins. In several of the examples given the evidence is inconclusive, but the examples are no less interesting for that.

Variation in endophytic fungi from roots and leaves of Lepanthes (Orchidaceae).

Little is known about non-mycorrhizal endophytic fungi in tropical orchids; still less is known about how endophytes vary within and between individual orchid plants. Fungal endophytes were isolated from roots and leaves of epiphytic and lithophytic orchids in the genus Lepanthes; seven species, from rainforests in Puerto Rico, were sampled. The endophytes observed most frequently were Xylaria species and Rhizoctonia-like fungi, found in 29% of roots and 19% of leaves, and 45 % of roots and 31 % of leaves, respectively. Five deuteromycete genera were also isolated, occurring in 19 % of roots and 43 % of leaves (combined). At least nine species of Xylaria were found, with several species sometimes occurring in a single plant. Differences between roots and leaves in frequency of Xylaria and Rhizoctonia isolates were not significant, although differences among orchid species in number and types of endophytes were. Heterogeneity of endophytes in single plants and plant organs was greater than differences between species. Many Lepanthes species are very restricted in distribution, and knowledge of their interactions with endophytes might be useful in species management.

Eutypa dieback in grapevines: differential production of acetylenic phenol metabolites by strains of Eutypa lata.

The production of acetylenic phenol metabolites in vitro by three strains of the ascomycete Eutypa lata, the causative agent of dying-arm disease in grapevines, has been investigated. Metabolite composition and yields differed significantly between strains and with growth medium but usually reached a maximum after 24-30 days of fungal growth. A general method for the analysis and identification of metabolites by gas chromatography/mass spectrometry of their trimethylsilyl ether derivatives was developed and individual compounds were quantitated by analytical high-performance liquid chromatography (HPLC) and separated by preparative HPLC. The phenolic aldehyde, eutypine (1), reported to be the grape phytotoxin, occurred in only one of the strains examined whereas the primary metabolite was the corresponding alcohol, eutypinol (2), the presumptive detoxification product. A novel metabolite was isolated as a major constituent, together with a minor component, and their structures were established by spectroscopic methods as a methoxyquinol, named eulatinol (4), and a chromene analog (9) of 2, respectively. The evidence suggests that 1 is not solely responsible for phytotoxicity in grapevines but that dying-arm disease may result from a suite of compounds elaborated by the fungus, with the composition dependent on fungal strain and nutritional source.

An assessment of natural product discovery from marine (sensu strictu) and marine-derived fungi.

The natural products community has been investigating secondary metabolites from marine fungi for several decades, but when one attempts to search for validated reports of new natural products from marine fungi, one encounters a literature saturated with reports from 'marine-derived' fungi. Of the 1000+ metabolites that have been characterized to date, only approximately 80 of these have been isolated from species from exclusively marine lineages. These metabolites are summarized here along with the lifestyle and habitats of their producing organisms. Furthermore, we address some of the reasons for the apparent disconnect between the stated objectives of discovering new chemistry from marine organisms and the apparent neglect of the truly exceptional obligate marine fungi. We also offer suggestions on how to reinvigorate enthusiasm for marine natural products discovery from fungi from exclusive marine lineages and highlight the need for critically assessing the role of apparently terrestrial fungi in the marine environment.

Sociomicrobiome of wood decay in a tropical rain forest: unraveling complexity.

Given that microbial interactions in nature are very complex, we propose that quorum-sensing, as well as quorum-quenching, phenazine and secondary metabolite production, resistance and toxin-antitoxin systems within a microbial community should all comprise the battery of processes involving the study of what we would define as the "sociomicrobiome". In the present study the genes/molecules, subsystems and taxonomic breakup of the mentioned processes were identified in decaying tropical wood from the El Yunque rainforest in Puerto Rico, and soil using a shotgun metagenomic approach. The rapid decomposition of wood and litter in tropical regions suggests that processes in these settings are governed by unexplored microbes with the potential of being further studied and exploited for various purposes. Both ecosystems were characterized by the presence of specific genes/molecules, subsystems and microbes associated with the mentioned processes, although the average abundances for specific processes differed. Of the sociomicrobiomes studied, that from El Yunque was found to be the most complex. The approach considered in the present study could also be applied to study the sociomicrobiome of other ecosystems.