Applying different infective structures of Beauveria bassiana to Coffea arabica infested with Hypothenemus hampei (Coleoptera: Curculionidae) under field

Coffee is the most appreciated beverage in worldwide; Brazil is the largest producer and exporter of this commodity. Organochlorine endosulfan was banned from the country in 2013 due to its teratogenic agent-related features. Since then, coffee plantations have experienced increased Hypothenemus hampei infestation rates. The aim of the current study is to assess variations in the rates of Coffea arabica fruits brocaded by H. hampei after the application of entomopathogenic fungal species Beauveria bassiana IBCB66. Experiments were carried out with 'Catuaí' and 'Mundo Novo' cultivars between 2018 and 2020, during the borer transit period. Three experiments were carried out based on the application of the aforementioned fungal species on the investigated coffee plant species, both by spraying and sprinkling, at 30-day intervals; 10 fruits were collected per face of each useful plant in each repetition. The experiment has followed a randomized blocks design with five treatments, including the control, and five repetitions, each. Beauveria bassiana Ecobass (IBCB66) wettable powder spray, at the concentration of 2 × 1013·ha­1, was used in experiments I and II. On the other hand, the mix used in experiment III was prepared with blastospores at concentration of 5 × 1012·ha­1 blastospores + 0.1% Silwet. The sprinkling process in all three experiments has used dry aerial conidia at concentration of 2 × 1013·ha­1. Collected data were subjected to analysis of variance (ANOVA), which was followed by Fisher's test at 5% probability level, in the SISVAR software. More than 35,000 fruits were assessed. In addition to variations between experiments, results have evidenced that the rate of brocaded fruits remained high.

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.

New species and records of Trichoderma isolated as mycoparasites and endophytes from cultivated and wild coffee in Africa.

A survey for species of the genus Trichoderma occurring as endophytes of Coffea, and as mycoparasites of coffee rusts (Hemileia), was undertaken in Africa; concentrating on Cameroon and Ethiopia. Ninety-four isolates of Trichoderma were obtained during this study: 76 as endophytes of healthy leaves, stems and berries and, 18 directly from colonized rust pustules. A phylogenetic analysis of all isolates used a combination of three genes: translation elongation factor-1α (tef1), rpb2 and cal for selected isolates. GCPSR criteria were used for the recognition of species; supported by morphological and cultural characters. The results reveal a previously unrecorded diversity of Trichoderma species endophytic in both wild and cultivated Coffea, and mycoparasitic on Hemileia rusts. Sixteen species were delimited, including four novel taxa which are described herein: T. botryosum, T. caeruloviride, T. lentissimum and T. pseudopyramidale. Two of these new species, T. botryosum and T. pseudopyramidale, constituted over 60% of the total isolations, predominantly from wild C. arabica in Ethiopian cloud forest. In sharp contrast, not a single isolate of Trichoderma was obtained using the same isolation protocol during a survey of coffee in four Brazilian states, suggesting the existence of a 'Trichoderma void' in the endophyte mycobiota of coffee outside of Africa. The potential use of these African Trichoderma isolates in classical biological control, either as endophytic bodyguards-to protect coffee plants from Hemileia vastatrix, the fungus causing coffee leaf rust (CLR)-or to reduce its impact through mycoparasitism, is discussed, with reference to the on-going CLR crisis in Central America.

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.

Evaluation of Terpene-Volatile Compounds Repellent to the Coffee Berry Borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae).

The coffee berry borer (CBB) is one of the main coffee pests in the world including Colombia. This pest is difficult to manage because of its cryptic habits and the continuous availability of coffee fruits. Among the new management strategies being tested is the use of volatile compounds as insect repellents. In this work, the behavioral response of female adult CBBs to terpenes previously identified in the CBB-repellent plant species Lantana camara was evaluated. α-Terpinene, (R)-limonene, farnesene and ß-caryophyllene terpenes were tested via a Y-tube olfactometer in which ripe coffee fruits were accompanied by terpenes at concentrations between 25 and 200 ppm. Only ß-caryophyllene induced a significant and consistent CBB repellent effect at all tested doses. The protective effect of microencapsulated ß-caryophyllene was then determined under laboratory conditions by incorporating the terpene in a colloidosome-gel system at 2.8 × 105 ng/h in the middle of coffee fruits with adult CBBs. The coffee fruits in turn presented a decrease in fruit infestation. Furthermore, the protection of coffee fruits when ß-caryophyllene gels were hung in coffee trees was evaluated in the field; infestations were artificially induced by the use of raisins (CBB-infested old coffee fruits) placed on the ground. Compared with unprotected trees, the trees treated with caryophyllene gels exhibited a 33 to 45% lower degree of infestation. Taken together, the results show that ß-caryophyllene is a promising compound for an integrated pest management (IPM) program in commercial coffee plantations.

Cyantraniliprole susceptibility baseline, resistance survey and control failure likelihood in the coffee berry borer Hypothenemus hampei.

Cyantraniliprole was recently registered for controlling the coffee berry borer Hypothenemus hampei, the main coffee pest in the world. In this study, baseline determination and resistance monitoring to cyantraniliprole were carried out in Brazilian populations of H. hampei. Evaluations were carried out for three years with representative field-collected populations from nine coffee-producing states in Brazil, using artificial diet containing the insecticide. The likelihood of control failure due to cyantraniliprole resistance was also determined. Populations from Campo do Meio, Linhares and Jaú were more susceptible (<2-fold resistance) to cyantraniliprole than populations from Patrocínio and Londrina (17-fold). Nonetheless, the frequency of cyantraniliprole resistance insects was low and not significant throughout the regions survey and the likelihood of control failure was negligible. Therefore, cyantraniliprole remains an important management tool against the coffee berry borer without current problems of control failure. However, enough field variation in susceptibility to cyantraniliprole exists justifying attention and careful management of this insecticide to prevent quick development of insecticide resistance in populations of this insect pest species.

Modelling the effect of temperature on the biology and demographic parameters of the African coffee white stem borer, Monochamus leuconotus (Pascoe) (Coleoptera: Cerambycidae).

The African coffee white stem borer Monochamus leuconotus (Pascoe) (Coleoptera: Cerambycidae) is a destructive insect pest of Arabica coffee trees in African highlands. Our study aims to provide information on the pest biology as influenced by temperature, determine thermal thresholds, and provide life table parameters for M. leuconotus reared in the laboratory. The life cycle of M. leuconotus was studied at seven constant temperatures in the range 15-35 °C, with 80 ± 5% RH and a photoperiod of L:D 12:12. Linear and nonlinear models were fitted to laboratory data to describe the impact of temperature on M. leuconotus development, mortality, fecundity and senescence. The complete life cycle was obtained between 18 and 30 °C, with the egg incubation period ranging 10.8-29.2 days. The development time was longest for the larva, with 194.2 days at 30 °C and 543.1 days at 18 °C. The minimum temperature threshold (Tmin) was estimated at 10.7, 10.0 and 11.5 °C, for egg, larva and pupa, respectively. The maximum temperature threshold (Tmax) was estimated at 37.4, 40.6 and 40.0 °C for egg, larva and pupa, respectively. The optimum temperature for immature stage survival was estimated between 23.0 and 23.9 °C. The highest fecundity was 97.8 eggs per female at 23 °C. Simulated life table parameters showed the highest net reproductive rate (Ro) of 11.8 daughters per female at 26 °C and maximal intrinsic rate of increase (rm) between 26 and 28 °C, with a value of 0.008. Our results will help understanding M. leuconotus biology as influenced by temperature and may be used to predict the distribution and infestation risk under climate warming for this critical coffee pest.

Differential Metabolic Responses Caused by the Most Important Insect Pest of Coffee Worldwide, the Coffee Berry Borer (Hypothenemus hampei).

The world's coffee supply is threatened by the coffee berry borer, Hypothenemus hampei, the most destructive pest affecting coffee production and quality. This study hypothesized that coffee berry borer infestation induces distinct metabolic responses in the green coffee seeds of Coffea arabica and Coffea canephora (robusta). A targeted metabolomics approach was conducted using liquid chromatography tandem mass spectrometry to quantify intracellular metabolites in infested and uninfested arabica and robusta green seeds. In parallel, the seed biomass content and composition were assessed for the same conditions. Coffee berry borer attack induced increases in the levels of chlorogenic acids in arabica seeds, whereas organic acids and sugar alcohols were more abundant in infested robusta seeds. Most importantly, a set of compounds was identified as biomarkers differentiating the metabolic response of these taxa to the coffee berry borer.

Functional response of Euseius concordis feeding on Oligonychus ilicis (Acari: Phytoseiidae, Tetranychidae).

Oligonychus ilicis (McGregor) (Acari: Tetranychidae), commonly known as the southern red mite, or as the coffee red spider mite in Brazil, is one of the main species of herbivorous mites that causes serious damage to coffee plants (Coffea spp.) and thus negatively affects coffee production. Among the biocontrol agents, predatory mites of the family Phytoseiidae play an important role in many biological control programs worldwide due to their potential as suppressor of mite populations mainly from the family Tetranychidae. One of the phytoseiid mites usually associated with O. ilicis is Euseius concordis (Chant), which often occurs abundantly in the coffee crops of Minas Gerais State, Brazil. This study was conducted to assess the predation potential of E. concordis feeding on the larvae and nymph stages of O. ilicis on coffee plants (Coffea arabica L.). Logistic regression analysis revealed a Holling type II functional response, showing that the number of O. ilicis killed by E. concordis increased gradually as the density of O. ilicis increased. Average daily oviposition also increased with prey densities above 6.3 mites/cm2, indicating that maximum oviposition rate is about 1 egg/day. Results of this study suggest that E. concordis has the potential to reduce O. ilicis populations, and this predatory mite can therefore be considered an important natural enemy of the pest O. ilicis in coffee plantations.

Temperature-dependent development and survival of immature stages of the coffee berry borer Hypothenemus hampei (Coleoptera: Curculionidae).

Although the coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae: Scolytinae) is the most destructive insect pest of coffee worldwide, there is much to learn about its thermal biology. This study aimed to develop temperature-based models for H. hampei development and to provide the thermal requirements of immature stages in the laboratory. Using a new observation method, larval development and survival were monitored daily on fresh Arabica coffee seeds, under seven constant temperatures in the range 15-35°C, with 80 ± 5% RH and 12:12 L:D photoperiod. Linear and non-linear functions were fitted to the development data plotted against temperature, using Insect Life Cycle Modelling software (ILCYM). Temperature significantly affected the development time of all immature stages. Egg incubation period ranged 4.6-16.8 days, under temperature between 30 and 15°C. No development occurred at 35°C and the larval stage did not develop to pupa at 15°C. The minimum temperature threshold (Tmin) estimated from linear regression was 10.5, 13.0, 15.0 and 13.0°C, for egg, larva, pupa and the total development from egg to adult, respectively. The maximum temperature threshold (Tmax) estimated from the Sharpe and DeMichele function was 32°C for egg to adult development. The thermal constant (k) was estimated at 78.1, 188.7, 36.5 and 312.5 degree days, for egg, larva, pupa and for egg to adult, respectively. Our results will help understand and predict the pest population dynamics and distribution in coffee plantations as impacted by temperature, and as such, will contribute to a more efficient management of the pest.

Revealing the respiratory system of the coffee berry borer (Hypothenemus hampei; Coleoptera: Curculionidae: Scolytinae) using micro-computed tomography.

The coffee berry borer (Hypothenemus hampei) is the most economically important insect pest of coffee globally. Micro-computed tomography (micro-CT) was used to reconstruct the respiratory system of this species for the first time; this is the smallest insect (ca. 2 mm long) for which this has been done to date. Anatomical details of the spiracles and tracheal tubes are described, images presented, and new terms introduced. The total volume and the relationship between tracheal lumen diameter, length and volume are also presented. The total length of the tracheal tubes are seventy times the length of the entire animal. Videos and a 3D model for use with mobile devices are included as supplementary information; these could be useful for future research and for teaching insect anatomy to students and the public in general.

Transcriptome and gene expression analysis of three developmental stages of the coffee berry borer, Hypothenemus hampei.

Coffee production is a global industry valued at approximately 173 billion US dollars. One of the main challenges facing coffee production is the management of the coffee berry borer (CBB), Hypothenemus hampei, which is considered the primary arthropod pest of coffee worldwide. Current control strategies are inefficient for CBB management. Although biotechnological alternatives, including RNA interference (RNAi), have been proposed in recent years to control insect pests, characterizing the genetics of the target pest is essential for the successful application of these emerging technologies. In this study, we employed RNA-seq to obtain the transcriptome of three developmental stages of the CBB (larva, female and male) to increase our understanding of the CBB life cycle in relation to molecular features. The CBB transcriptome was sequenced using Illumina Hiseq and assembled de novo. Differential gene expression analysis was performed across the developmental stages. The final assembly produced 29,434 unigenes, of which 4,664 transcripts were differentially expressed. Genes linked to crucial physiological functions, such as digestion and detoxification, were determined to be tightly regulated between the reproductive and nonreproductive stages of CBB. The data obtained in this study help to elucidate the critical roles that several genes play as regulatory elements in CBB development.

Coffee berry borer (Hypothenemus hampei) (Coleoptera: Curculionidae) development across an elevational gradient on Hawai'i Island: Applying laboratory degree-day predictions to natural field populations.

Coffee berry borer (CBB, Hypothenemus hampei) (Coleoptera: Curculionidae: Scolytinae) is the most destructive pest of coffee worldwide. Information on CBB development times can be used to predict the initiation of new infestation cycles early in the coffee-growing season and thus inform the timing of insecticide applications. While laboratory estimates of CBB development under constant conditions exist, they have not been applied under the heterogeneous environmental conditions that characterize many coffee-growing regions. We measured CBB development times and abundance in commercial coffee farms across an elevational gradient on Hawai'i Island and applied thermal accumulation models from previous laboratory studies to test their fit to field data. Artificial lures were used to infest coffee berries at five farms ranging in elevation from 279-792 m, and weather variables were monitored at macro (farm-level) and micro (branch-level) scales. CBB development was followed in the field from the time of initial berry infestation by the founding female through the development of F1 mature adults. Mean development time from egg to adult across all sites was 38.5 ± 3.46 days, while the mean time required for the completion of a full life cycle (from time of infestation to presence of mature F1 females) was 50.9 ± 3.35 days. Development time increased with increasing elevation and decreasing temperature. Using macro-scale temperature data and two different estimates for the lower temperature threshold (14.9°C and 13.9°C), we estimated a mean requirement of 332 ± 14 degree-days and 386 ± 16 degree-days, respectively, from the time of berry infestation to the initiation of a new reproductive cycle in mature coffee berries. Similar estimates were obtained using micro-scale temperature data, indicating that macro-scale temperature monitoring is sufficient for life-cycle prediction. We also present a model relating elevation to number of CBB generations per month. Our findings suggest that CBB development times from laboratory studies are generally applicable to field conditions on Hawai'i Island and can be used as a decision support tool to improve IPM strategies for this worldwide pest of coffee.

Hysteresis and critical transitions in a coffee agroecosystem.

Seeking to employ ecological principles in agricultural management, a classical ecological debate provides a useful framing. Whether ecosystems are controlled from above (predators are the limiting force over herbivores) or from below (overutilization of plant resources is the limiting force over herbivores) is a debate that has motivated much research. The dichotomous nature of the debate (above or below) has been criticized as too limiting, especially in light of contemporary appreciation of ecological complexity-control is more likely from a panoply of direct and indirect interactions. In the context of the agroecosystem, regulation is assumed to be from above and pests are controlled, a way of using ecological insights in service of an essential ecosystem service-pest control. However, this obvious resolution of the old debate does not negate the deeper appreciation of complexity-the natural enemies themselves constitute a complex system. Here we use some key concepts from complexity science to interrogate the natural functioning of pest regulation through spatially explicit dynamics of a predator and a disease operating simultaneously but distributed in space. Using the green coffee scale insect as a focal species, we argue that certain key ideas of complexity science shed light on how that system operates. In particular, a hysteretic pattern associated with distance to a keystone ant is evident.

Meloidogyne daklakensis n. sp. (Nematoda: Meloidogynidae), a new root-knot nematode associated with Robusta coffee (Coffea canephora Pierre ex A. Froehner) in the Western Highlands, Vietnam.

The root-knot nematode species Meloidogyne daklakensis n. sp. was discovered on the roots of Robusta coffee (Coffea canephora Pierre ex A. Froehner) in Dak Lak Province, Vietnam. This species is characterized by the females having rounded or oval perineal patterns, smooth, regular, continuous striae, and reduced lateral lines. The dorsal arch is low, rounded and encloses a quite distinct vulva and tail tip. The stylet is normally straight with well-developed and posteriorly sloped knobs. The males have a rounded cap that extends posteriorly into the lip region. The procorpus is outlined distinctly, and is three times longer than the metacorpus. The metacorpus is ovoid, with a strong valve apparatus. The species closely resembles M. marylandi, M. naasi, M. ovalis, M. panyuensis, M. lopezi, M. mali and M. baetica in the perineal pattern of the females, and the morphology of the males and the second-stage juveniles. Nonetheless, it can be differentiated from other species by a combination of morphometric, morphological and molecular characteristics. Phylogenetic analysis was conducted based on the internal transcribed spacer (ITS) and 28S rDNA as well as the region between the cytochrome c oxidase I (COI) and cytochrome c oxidase II (COII) mitochondrial genes. Herein, this nematode is described, illustrated, and designated as a new species, Meloidogyne daklakensis sp. n., based on morphometric, morphological and molecular analyses.

An atoxigenic L-strain of Aspergillus flavus (Eurotiales: Trichocomaceae) is pathogenic to the coffee twig borer, Xylosandrus compactus (Coleoptera: Curculionidea: Scolytinae).

This study isolated and evaluated virulence of fungal entomopathogens of Xylosandrus compactus - an important pest of Robusta coffee in Sub-Saharan Africa. A survey was conducted in five farming systems in Uganda to isolate entomopathogens associated with X. compactus. Four fungal isolates were screened for virulence against X. compactus in the laboratory at 1 × 107 conidia ml-1 where an atoxigenic L-strain of A. flavus killed 70%-100% of all stages of X. compactus compared with other unidentified isolates which caused 20%-70% mortalities. The time taken by A. flavus to kill 50% of X. compactus eggs, larvae, pupae and adults in the laboratory was 2-3 days; whereas the other unidentified fungal isolates took 4-7 days. The concentrations of A. flavus that killed 50% of different stages of X. compactus were 5 × 105 , 12 × 105 , 17 × 105 and 30 × 105 conidia ml-1 for larvae, eggs, pupae and adults respectively. A formulation of A. flavus in oil caused higher mortalities of X. compactus larvae, pupae and adults in the field (71%-79%) than its formulation in water (33%-47%). The atoxigenic strain of A. flavus could therefore be developed into a safe biopesticide against X. compactus.

Analysis of volatile profiles of green Hawai'ian coffee beans damaged by the coffee berry borer (Hypothenemus hampei).

BACKGROUND: The coffee berry borer (CBB), Hypothenemus hampei, is the most destructive insect pest of coffee globally, causing significant losses in yield and leading to 'off' flavors in damaged beans. Automated headspace sampling (AHS) and gas chromatography-mass spectrometry (GC-MS) were used to investigate changes in the volatile profiles of CBB-damaged green coffee beans. Green coffee from three coffee farms on the island of Hawai'i were sorted into three levels of CBB damage: non-damaged, slightly damaged (1-2 pinholes/bean), and heavily damaged (> 2 pinholes/bean). RESULTS: Distinct differences were found between green coffee bean samples based on the amounts of eight prominent volatiles. The amount of CBB damage was particularly correlated with the amount of both hexanal and 2-pentylfuran. Principal component analysis showed clustering of non-damaged green beans, which did not overlap with the slightly or heavily damaged clusters. Good separation was also found between a mixture of 50% slightly damaged and non-damaged coffee. However, 20% slightly damaged and non-damaged coffee clusters showed strong overlap. CONCLUSION: Understanding the effects of CBB damage on coffee flavor profiles is critical to quality control for this valuable agricultural product. The results of this study show that the volatile profiles of green coffee beans vary with CBB damage. With specific volatile profiles for CBB-damaged coffee identified, coffee samples can be tested in the lab, or potentially on the farm or in coffee mills, to identify high levels of CBB damage that may lead to off flavors and a reduction in product quality and value. © 2018 Society of Chemical Industry.

High diversity and variability in the bacterial microbiota of the coffee berry borer (Coleoptera: Curculionidae), with emphasis on Wolbachia.

AIMS: Variation in microbiota of the coffee berry borer (CBB) Hypothenemus hampei was studied. Diversity, structure and function of bacterial communities were compared between eggs vs adults, CBBs from shade coffee vs sun coffee, CBBs from the field vs raised in the laboratory, and CBBs with and without the antibiotic tetracycline. METHODS AND RESULTS: We sequenced the region V4 of the gene 16 S rRNA. Pseudomonadaceae and Enterobacteriaceae, particularly Pseudomonas and Pantoea, dominated microbiotas of the CBB. Comparative functional inferences with PICRUSt suggested that samples from the field were enriched for genes involved in carbohydrate and protein digestion and absorption, while laboratory-reared samples were higher in genes for melanization and caffeine metabolism. CONCLUSIONS: Microbiotas of the CBB were diverse and dominated by the genus Pseudomonas, several species of which have been previously associated with caffeine degradation in this insect. Wolbachia was the only endosymbiont detected with known ability to manipulate host reproduction. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that stage of development and origin of samples affected the structure and function of the CBB's bacterial communities. This is the first attempt to predict functional significance of the CBB microbiota in nutrition, reproduction and defence.

Integrative taxonomy of root-knot nematodes reveals multiple independent origins of mitotic parthenogenesis.

During sampling of several Coffea arabica plantations in Tanzania severe root galling, caused by a root-knot nematode was observed. From pure cultures, morphology and morphometrics of juveniles and females matched perfectly with Meloidogyne africana, whereas morphology of the males matched identically with those of Meloidogyne decalineata. Based on their Cox1 sequence, however, the recovered juveniles, females and males were confirmed to belong to the same species, creating a taxonomic conundrum. Adding further to this puzzle, re-examination of M. oteifae type material showed insufficient morphological evidence to maintain its status as a separate species. Consequently, M. decalineata and M. oteifae are synonymized with M. africana, which is herewith redescribed based on results of light and scanning electron microscopy, ribosomal and mitochondrial DNA sequences, isozyme electrophoresis, along with bionomic and cytogenetic features. Multi-gene phylogenetic analysis placed M. africana outside of the three major clades, together with M. coffeicola, M. ichinohei and M. camelliae. This phylogenetic position was confirmed by several morphological features, including cellular structure of the spermatheca, egg mass position, perineal pattern and head shape. Moreover, M. africana was found to be a polyphagous species, demonstrating that "early-branching" Meloidogyne spp. are not as oligophagous as had previously been assumed. Cytogenetic information indicates M. africana (2n = 21) and M. ardenensis (2n = 51-54) to be a triploid mitotic parthenogenetic species, revealing at least four independent origins of mitotic parthenogenesis within the genus Meloidogyne. Furthermore, M. mali (n = 12) was found to reproduce by amphimixis, indicating that amphimictic species with a limited number of chromosomes are widespread in the genus, potentially reflecting the ancestral state of the genus. The wide variation in chromosome numbers and associated changes in reproduction modes indicate that cytogenetic evolution played a crucial role in the speciation of root-knot nematodes and plant-parasitic nematodes in general.

Identification of microsatellite markers in coffee associated with resistance to Meloidogyne exigua.

Meloidogyne species are destructive phytonematodes that result in reduced yields of coffee. The classic test for resistance to Meloidogyne exigua in coffee progenies is both expensive and time-consuming. The use of molecular marker techniques can assist the selection process when it is difficult to measure the phenotype, such as in cases of resistance to nematode infestation. The objective of this study was to identify microsatellite markers associated with resistance to M. exigua in F5 progenies of coffee derived from a cross between Híbrido de Timor 440-10 and Catuaí Amarelo IAC 86. Of the 44 simple sequence repeat (SSR) markers evaluated, 11 showed a polymorphic pattern with a mean number of 4.5 alleles per marker. Clustering analysis classified 82 progenies into three groups related to the response to nematodes and parental genotypes allocated to different groups (resistant and susceptible). SSRCafé 40 allele 2, SSRCafé 15 allele 3, SSRCafé 20 allele 3, and SSRCafé 13 allele 1 were negatively correlated with reproduction factor. In addition, SSRCafé 13 allele 2, SSRCafé 19 allele 3, SSRCafé 40 allele 2, SSRCafé 15 allele 3, and SSRCafé 20 allele 3 were correlated with the root gall index of M. exigua. These SSR markers, which have been validated in this population, represent a potential method to select progenies resistant to nematodes in coffee-breeding programs.