Isolation,morphological and molecular characterization of Bacillus thuringiensis strains against Hypothenemus hampei Ferrari (Coleoptera: Curculionidae: Scolytinae)

The coffee berry borer Hypothenemus hampei Ferrari, 1876 (Coleoptera: Curculionidae: Scolytinae) is considered the most serious pest of the coffee crop and is controlled primarily with the use of chemical insecticides. An alternative to this control method is the use of the entomopathogenic bacterium, Bacillus thuringiensis Berliner, 1911. Therefore, the objective of this work was to select strains of B. thuringiensis virulent against H. hampei and characterize them by morphological and molecular methods to identify possible genes for the production of genetically modified plants. To achieve this objective, 34 strains of B. thuringiensis underwent a selective bioassay to evaluate their toxicity to H. hampei first-instar larvae. Among the strains tested, 11 and the standard B. thuringiensis subspecies israelensis (IPS-82) caused mortality above 90%. Then, the median lethal concentration (LC₅₀) was estimated for these strains followed by characterization using morphological, biochemical and molecular methods. The lowest LC₅₀ was obtained for strain BR58, although this concentration did not differ significantly from that of the standard strain IPS-82 or from that of strains BR137, BR80 and BR67. The molecular characterization detected cry4A, cry4B, cry10, cry11 and cyt1 genes in 10 of the most virulent strains (BR58, BR137, BR80, BR81, BR147, BR135, BR146, BR138, BR139, BR140). Strain BR67 differed completely from the others and amplified only the cry3 gene. This strain was more virulent than BR135, BR146, BR138, BR139 and BR140, but it did not differ from BR58, BR137, BR80, BR81 and BR147. The protein profile revealed proteins of 28, 65, 70 and 130 kDa, and the morphological analysis identified spherical crystalline inclusions in all strains. The results showed that the 11 strains studied have potential for use as a gene source for insertion into coffee plants for the control H. hampei, especially the cry3, cry4A, cry4B, cry10, cry11 and cyt1 genes, that were repeated in the most virulent isolates.     

Natural occurrence of Beauveria bassiana in Hypothenemus hampei (Coleoptera: Curculionidae) populations in unsprayed coffee fields

Three unsprayed coffee farms (farm 1, 2 and 3) were studied for the natural occurrence of the insect pathogenic fungus Beauveria bassiana in Hypothenemus hampei populations throughout the rainy season of 2004 (July-November) and 2005 (July-December). B. bassiana infections were found during most sampling dates in both years, on all three farms. The B. bassiana infection levels were higher in 2005 than in 2004 with mean prevalence of 12.1% and 2.7%, respectively. No consistent significant differences in infection level between farms were found in any of the years. B. bassiana infection levels fluctuated widely throughout the season, and peaked at 13.5% on farm 3 in 2004 and at 44.0% on farm 1 in 2005. The H. hampei population was significantly higher in 2004 than in 2005, with 6.9% of the berries infested in 2004 and only 0.7% in 2005. In both years, the H. hampei infestation level was significantly higher on farm 2. No consistent significant differences in H. hampei infestation levels were found between sampling dates on any of the farms. H. hampei infestation levels fluctuated throughout both seasons, and peaked at 15.3% on farm 2 in 2004 and 2.2% on farm 2 in 2005. No consistent density dependent correlation between H. hampei infestation level and B. bassiana infection level was found. Correlations between climatic conditions and B. bassiana or H. hampei were not found.     

Bacillus thuringiensis serovar israelensis is highly toxic to the coffee berry borer, Hypothenemus hampei Ferr. (Coleoptera: Scolytidae)

A native collection of Bacillus thuringiensis strains was screened, once a reliable bioassay technique to assess the toxicity against the coffee berry borer (CBB) first-instar larvae was developed. A first round of bioassays with 170 strains indicated that the great majority of them showed no or very little insecticidal activity and that very few showed significant levels of toxicity. Interestingly, only those strains that had previously been associated with mosquitocidal activity were also toxic to CBB. Qualitative bioassays (using one high dose) were carried out only with those native mosquitocidal strains, corroborating their significant toxicity towards the CBB first-instar larvae. Most of these strains belong to serovar israelensis. In a second approach, strains from the Institut Pasteur type collection, whose mosquitocidal activity had been previously demonstrated, were also subjected to bioassays. Only those strains that showed a comparable protein content in their parasporal crystals to the israelensis type strain also showed high levels of toxicity towards CBB. Finally, an accurate LC(50) was estimated, using purified parasporal crystals from B. thuringiensis serovar israelensis type strain, at 219.5 ng cm(-2) of diet. All the statistical requirements for a reliable estimator were fulfilled. This is the first report of B. thuringiensis serovar israelensis being active against a coleopteran species.     

Increased pathogenicity against coffee berry borer, Hypothenemus hampei (Coleoptera: Curculionidae) by Metarhizium anisopliae expressing the scorpion toxin (AaIT) gene

Coffee berry borer (CBB) is the Worlds most devastating coffee pest causing an estimated US$500 million worth of losses annually through damage and control costs. Beauveria bassiana and Metarhizium anisopliae have been employed to control this pest but their low virulence (slow kill and large inoculums) is an important factor constraining their use. M. anisopliae (AaIT-Ma549) has been modified to express the scorpion toxin (AaIT) in insect hemolymph and this greatly increased pathogenicity against Manduca sexta and Aedes aegypti. Here, we demonstrate that AaIT-Ma549 was also dramatically more virulent against CBB, and we provide a much more comprehensive analysis of infection processes and post-mortality development than in the previous research. We evaluated several spore concentrations (10¹ through 10⁷ spores/ml) of both the wild type and recombinant strain. At concentrations of 10¹, 10² and 10³ spores/ml, the recombinant strain significantly increased mortality of CBB by 32.2%, 56.6% and 24.6%, respectively. The medial lethal concentration (LC₅₀) was reduced 15.7-fold and the average survival time (AST) was reduced by 20.1% to 2.98 ± 0.1 days with 10⁷ spores/ml. This is the first occasion that an entomopathogenic fungus has been found to kill CBB in less than 3 days. However, AaIT-Ma549 produces significantly fewer spores on cadavers than the parental strain.     

Phylogenetic origins of African and Neotropical Beauveria bassiana s.l. pathogens of the coffee berry borer, Hypothenemus hampei

A phylogenetic epidemiological study of Beauveria bassiana s.l. was conducted for African and Neotropical pathogens of the coffee berry borer (CBB), Hypothenemus hampei, based on inferences from two nuclear intergenic regions, EFutr and Bloc. CBB pathogens were distributed among four terminal clades, however, the majority of African and Neotropical isolates cluster in a well-supported monophyletic group, informally designated AFNEO_1. Although the relationship between African and Neotropical AFNEO_1 is unresolved, the majority of alleles detected were exclusive to either the African or the Neotropical populations. These fixed genetic differences suggest that their disjunction predates the world trade in coffee. Neotropical AFNEO_1 have a broad host range and CBB pathogens are intermixed phylogenetically with isolates from diverse indigenous insects. Several Neotropical AFNEO_1 isolates were isolated from coffee plants as epiphytes or endophytes, thus plants themselves may potentially serve as reservoirs of pathogens against their insect pests. Topological incongruence between the EFutr and Bloc phylogenies of Neotropical AFNEO_1 may signify that individuals within this population are recombining.     

Efficacy of Beauveria bassiana applications on coffee berry borer across an elevation gradient in Hawaii

ABSTRACT

The effect of three rates of a commercial formulation of Beauveria bassiana Strain GHA was evaluated against the coffee berry borer (CBB) Hypothenemus hampei Ferrari (Coleoptera: Curculionidae: Scolytinae), at three commercial coffee farms located at different altitudes on the island of Hawaii. H. hampei infestation and natural prevalence of B. bassiana increased with the elevation. At 145 metres above sea level (Farm 1), beetle infestation was 3.9%; at 538?m (Farm 2), beetle infestation was 12.2%; and at 768?m (Farm 3) infestation was 22.3%. The prevalence of natural B. bassiana killing CBB was 5.5% on Farm 1, 3.3% on Farm 2 and 23.1% on Farm 3. Monthly applications of B. bassiana resulted in no significant differences in levels of CBB infestation among treatments. Similarly, rates of infested berries with visually detectable signs of B. bassiana were similar among the B. bassiana treatments, ranging from 0.44% to 4.24%, and those percentages were larger than the treatments without B. bassiana. The percentage of females killed by Beauveria ranged from 69% to 95%. Effect of dose of BotaniGard^® ES was reduced when beetles were in C position compared to A and B positions. B. bassiana can be an important component of an integrated pest management program for CBB.     

Optimization of coffee berry borer, Hypothenemus hampei Ferrari (Col., Scolytidae), mass trapping with an attractant mixture

Mass trapping is a technique currently being considered to control the coffee berry borer (CBB), Hypothenemus hampei Ferrari (Col., Scolytidae), as part of the integrated management of its populations. However, the research undertaken in this field has variable and sometimes contradictory results. The purpose of this study carried out with a single type of trap (experimental model '1B') was to evaluate the technique and define parameters for its efficient use. The first result obtained was detection of the excellent attractant properties of the ethanol–methanol mixture, and its efficiency related to the proportion of the two alcohols. Coffee borers were not attracted to traps with caffeine, green coffee powder, freshly pulped and hulled coffee berries and ground CBB. It was shown that the ethanol–methanol mixture (1 : 1) release rate varied without affecting the capture rate, and that the red colour of the trap substantially increased CBB attraction. In terms of trap set-up, it was found that capture rates were three times better with a trap height at 1.2 m, than that for a position near ground level. In addition, the best density of traps for achieving efficient mass trapping was 22 units per hectare. These results show the importance of developing an attractant using pure compounds, and of improving the trap and the trapping technique in line with CBB behaviour.     

Beauveria bassiana -A Potential Mycopesticide for the Efficient Control of Coffee Berry Borer, Hypothenemus hampei (Ferrari) in India

The Coffee Berry Borer (CBB), Hypothenemus hampei (Ferrari) (Coleoptera: Scolytidae) has been a serious insect pest of coffee cultivars C. robusta and C. catimor in India since 1991, causing 40-80% coffee bean loss. To combat this important pest, an indigenous entomopathogenic fungus Beauveria bassiana (Balsamo) Vuillemin was isolated from dead and moribund coffee berry borers from the wild. The fungus was cultured on yeast extractpeptone supplemented liquid medium. The mycelial mat was harvested from 12-day old cultures and lyophilized. A suspension of the most virulent isolate (Bb2) was prepared in sterile water and used as a mycopesticide. The laboratory studies were conducted on coffee berry borers by applying conidial suspensions at a dosage rate of 1 ×10 6 conidia ml -1 . Pest mortality with the Bb2 isolate increased from 69.3% to 95.3% with an increase in relative humidity (RH) at 25 ±2°C. Field experiments were conducted in a coffee plantation area in the Kodagu district of Karnataka, and the results showed that, under favourable environmental conditions (27- 29°C; 82-91% RH; 10-15 inches rainfall per year), the fungus required only eight days to colonize and kill the target pest. A maximum of 75.6% insect mortality was recorded 24 days after spraying. Large-scale field trials conducted in five plantation plots between September 1995 and September 1998 showed significant insect mortality both in C. robusta and C. catimor cultivars of coffee. The potential use of this indigenous fungal strain of B. bassiana as a mycopesticide for management of CBB in India is discussed.     

Evaluation of Beauveria bassiana (Ascomycota: Hypocreales) as a control of the coffee berry borer Hypothenemus hampei (Coleoptera: Curculionidae: Scolytinae) emerging from fallen,infested coffee berries on the ground

The effects of Beauveria bassiana strains on coffee berry borers (CBB), which emerge from infested berries left on soil, and its impact on the infestation of coffee berries on tree branches were evaluated at two Experimental Stations (Naranjal-Caldas and Paraguaicito-Quindio) in the Colombian coffee zone. Using a completely randomized design with 10 repetitions, 50 coffee berries artificially infested with CBB were placed on the base of a coffee tree. Four treatments including B. bassiana strain Bb9205, a mixture of Cenicafé strains (Bb9001, Bb9024 and Bb9119), a commercial formulation of B. bassiana and a control (water) were sprayed with 1×10⁹ conidia per tree. After 30 days, all fungal strains lowered the infestation levels of the coffee berries on the trees at both locations. The mixture of Cenicafé strains decreased the tree infestation between 50 and 30% at both locations. In the berries dissected from each treated tree, insect mortality was about 40% at both locations compared to 15% in the control. B. bassiana strains also decreased the insect population inside the newly infested berries on the trees by 55–75%. The mixture of Cenicafé strains was the most effective for decreasing insect populations. B. bassiana significantly decreased CBB populations that emerged from fallen, infested, coffee berries and reduced future insect generations.     

Artificial diet sandwich reveals subsocial behaviour in the coffee berry borer Hypothenemus hampei (Coleoptera: Curculionidae: Scolytinae)

An artificial diet sandwich, consisting of coffee berry borer artificial diet within two glass plates, has been developed to elucidate the behaviour of the coffee berry borer, an insect that in nature spends most of its life cycle inside the coffee berry. Various types of behaviour have been observed for the first time, including gallery construction, oviposition, gallery blocking, mating and most remarkably, subsocial tasks such as maternal sanitation and tending of eggs and larvae. This observational technique is a breakthrough for studies and manipulations of the coffee berry borer's social behaviour and could be applicable to other bark beetles, consequently yielding important insights into the origin of parental care in scolytine beetles.     

Coffee berry borer Hypothenemus hampei (Coleoptera: Curculionidae): searching for sustainable control strategies

The coffee berry borer Hypothenemus hampei (Ferrari) is the most serious pest of the world's most valuable tropical export crop. Since the last review on this insect was published six years ago, many new studies have contributed to an improved insight into the biology and ecology of the beetle, and have indicated new avenues for integrated and biological control. The latest developments in research, both laboratory and field, on the pest, its natural enemies and their implications for integrated control of H. hampei are summarized, with a particular focus on the situation in The Americas. Lately, the global coffee industry has changed radically; it has suffered a long cycle of lowest-ever world market prices caused by overproduction and technological change. At the same time, the advent of sustainable certification schemes has had a major impact on the industry. The role of integrated pest management and biological control of H. hampei in an era of changes in the coffee industry is discussed.     

Interactions among bethylid parasitoid species attacking the coffee berry borer,Hypothenemus hampei (Coleoptera: Scolytidae)

The question of whether biological control is most likely achieved by deploying single or multiple species of biological control agents is much debated. While utilizing several natural enemies may enhance control, there is also the potential for disruptive inter-specific interactions. Such interactions may be studied in the laboratory by focusing on the details of the interactions themselves and attempting to infer population level consequences from their sum, or by focusing more directly on the overall effects on natural enemy populations: we term these approaches ‘reductionist’ and ‘holistic.’ Here we conduct a holistic laboratory study on interactions between three species of parasitoid wasps that are parasitoids of the coffee berry borer, Hypothenemus hampei (Ferrari) (Coleoptera: Scolytidae): Cephalonomia stephanoderis Betrem, C. hyalinipennis Ashmead and Prorops nasuta Waterston (all Hymenoptera: Bethylidae). We find evidence for both intra- and inter-specific resource competition. Interactions between C. stephanoderis and P. nasuta, both indigenous to Africa, appear to be approximately symmetrical, while C. hyalinipennis, naturally found in the coffee plantations of Chiapas, Mexico, may exert a disruptive influence. C. hyalinipennis also has a low population growth rate. We now consider it to be a detrimental invader of the Mexican coffee agro-ecosystem that should not be encouraged by augmentative release or introduced into other regions. Overall, the most successful species, in terms of both emergence and female production, was P. nasuta. We compare these results with those from prior reductionist and holistic studies, and with observations on patterns of establishment of these bethylid species in the field. Given that it is increasingly clear that disruptive inter-specific interactions are generally common when multiple species are deployed in biological control, screening of potential agents should consider such interactions alongside the more ‘traditional’ focus on host specificity.     

Biological control of the coffee berry borer Hypothenemus hampei (Coleoptera: Curculionidae) by Phymastichus coffea (Hymenoptera: Eulophidae) in Colombia

The potential of the eulophid parasitoid Phymastichus coffea LaSalle to control coffee berry borer Hypothenemus hampei (Ferrari) populations under field conditions in Colombia was evaluated. Parasitoid adults were released one, five and nine days after artificial infestations of 90-, 150- and 210-day-old coffee berries with H. hampei females. The position of the beetle inside the berry and the parasitism levels were assessed ten days after each P. coffea release. Parasitism of H. hampei by P. coffea was significantly affected by the age of the berries at the time of infestation, and by the position of the beetle inside the berries. Highest levels of parasitism were recorded in 150-day-old berries (75-85%) and in 90-day-old berries (75%) when P. coffea were released one day after the artificial infestation with H. hampei. In 150-day-old berries, highest levels of parasitism were recorded for H. hampei found in the outer layer of the endosperm followed by beetles penetrating the exocarp. Increasing the time of P. coffea releases after the artificial infestations with H. hampei led to decreased levels of parasitism in beetles attacking 90- and 150-day-old coffee berries. Low levels of parasitism were recorded in H. hampei females infesting older coffee berries because most of the beetles had already constructed galleries deep in the endosperm of the berries, i.e. out of reach of the parasitoid. The potential of P. coffea for biological control of coffee berry borer in Colombia is discussed.     

Sterility of the coffee berry borer,Hypothenemus hampei (Coleoptera: Curculionidae), caused by the nematode Metaparasitylenchus hypothenemi (Tylenchidae: Allantonematidae)

Metaparasitylenchus hypothenemi is an endoparasitic nematode that causes partial or total sterility of coffee berry borer (Hypothenemus hampei) females, although the causes are unknown. Fecundity and the average size of the common and lateral oviduct, vitellarium, and germarium in the four ovarioles (I, II, III and IV) were compared between parasitised and non-parasitised insects to determine the causes of sterility. The nematode significantly lowers the number of oocytes and 86% of parasitised insects (24 out of 28 insects) were sterile, while fecundity in the remaining 13% was non-significantly different to that in non-parasitised insects. No significant differences were recorded in the size of the common oviduct, lateral oviduct, vitellarium, and germarium between parasitised and non-parasitised insects and the nematode does not cause any apparent damage on the surface of the ovary.     

Potential of Metarhizium anisopliae and Beauveria bassiana to control Dinoderus porcellus (Coleoptera: Bostrychidae) infesting yam chips

The beetle Dinoderus porcellus Lesne is a serious storage insect pest that causes important losses by destroying stocks of yam chips. In the aim to found an alternative control method to the use of synthetic insecticides for its management, the virulence of the entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin (isolate Bb115) and Metarhizium anisopliae (Metschnikoff) Sorokin (isolate Met 31) against adults of D. porcellus was evaluated under laboratory conditions (25 ± 2 °C and 70 ± 5% RH). Then, the effectiveness of the most virulent entomopathogenic fungus as biological agent against D. porcellus was assessed under farmer storage conditions. For each entomopathogenic fungus isolate, four conidial concentration (0, 10⁵, 10⁷, and 10⁹ conidia/mL) at the dose of 1 µL were inoculated topically on D. porcellus adults (3–5 days old). Observations focused on insect mortality, cadaver sporulation and weight loss of yam chips. Lethal dose and lethal time values were estimated using probit analysis. Both fungal isolates at all conidial dose caused more than 50% mortality on day 7, with the highest mortality (94.44%) achieved using B. bassiana at the 10⁹ conidia/mL. LT₅₀ values for B. bassiana and M. anisopliae isolates were 2.63 and 3.35 days, respectively, while their LT₉₀ values were 6.15 and 9.87 days, respectively. Yielding the lower LD₉₀ values and the highest rates of cadaver sporulation, B. bassiana isolate appeared as the most virulent against D. porcellus. After 3 months of storage, comparatively to the control, the B. bassiana isolate at the highest conidial dose (10⁹ conidia/mL) significantly reduced D. porcellus populations, and weight loss of yam chips. This study revealed the potential of B. bassiana and M. anisoplae isolates as biological control agent against D. porcellus for yam chips protection.     

Responses of coffee berry borer, Hypothenemus hampei (Ferrari)(Coleoptera: Scolytidae), to vertical distribution of methanol: ethanol traps

Captures of the coffee berry borer (CBB) Hypothenemus hampei (Ferrari) were assessed in traps in the field. IAPAR designed traps [plastic bottles (2 L) lured with methanol:ethanol (1:1) in a vessel] were placed either at 0.5, 1.0 and 1.5m high from the ground or simultaneously tested in the 2004 fructification season. Traps placed at the three heights trapped 5.5 times more CBB than the others, mostly at the traps placed at 0.5 m (75%). Treatments using the IAPAR designed trap placed at 1.2 m high; IAPAR trap with a white plastic plate above (IAPAR modified I) at 1.2 m high; IAPAR at 0.5 m high and two additional vessels at 1.0 and 1.5m high (IAPAR modified II) and T-163 trap [three red plastic cups (300 ml) and a red plastic plate as a cover] lured with M:E (1:1) at 1.2m height were compared in the vegetative (2005) and fructification (2006) periods. IAPAR modified II (dispenser vessels placed at 0.5, 1.0 and 1.5 m) trapped more beetles than the remaining types (2.72 times more beetles than IAPAR design); and IAPAR modified I traps trapped more beetles than T 163 and IAPAR traps in the vegetative period. In the reproductive period, IAPAR modified II trapped less beetles than IAPAR and IAPAR modified I. In 2007 vegetative season, IAPAR modified II trap were compared with IAPAR trap and trapped 2.8 times more beetles. The positive responses to a vertical distribution of the volatile attractants in the vegetative period of the planting allow the development of more efficient trapping systems for CBB.     

Development of an improved laboratory production technique for the coffee berry borer Hypothenemus hampei, using fresh coffee berries

The suitability of a mixture of plaster of Paris and charcoal as a means to regulate the moisture content of coffee berries and the relative humidity (moisture conditions) of the rearing environment and its impact on rearing the coffee berry borer (CBB), Hypothenemus hampei (Ferrari) (Coleoptera: Curculionidae), was evaluated under laboratory conditions using two types of coffee. Coffee berries were kept individually in vials with a 1-cm layer of the mixture, and the fresh weight of the berries was assessed, as well as the penetration of CBB into the berries, its survival, and its progeny production over a period of 55 days. Significantly higher survival and progeny production was achieved when using the mixture regardless of the coffee type. Compared to the vials without plaster of Paris/charcoal, a six- to sevenfold increase in survivorship of the F1 was recorded when using plaster of Paris/charcoal and in the latter treatment berries harboured on average more than 100 individuals, whereas only 1.7 in the vials without plaster of Paris.     

A review of the biology and control of the coffee berry borer, Hypothenemus hampei (Coleoptera: Scolytidae)

The coffee berry borer, Hypothenemus hampei Ferrari, is a serious problem for the majority of the world's coffee growers and has proved to be one of the most intractable of present day pests. Despite a great deal of research, control still depends largely on the application of the organochlorine insecticide endosulfan, which is damaging to the environment, or a series of cultural and biological control methods which give variable and unpredictable results. This review summarizes the most important aspects of the biology and ecology of H. hampei and its control and identifies weak points in the knowledge about this pest. Emphasis is placed upon an analysis of the non-chemical control methods available and suggestions are offered for novel ecological and environmental factors worthy of further research, in the search for viable and sustainable control methods.     

Altitude and coffee production systems influence extent of infestation and bean damage by the coffee berry borer

The coffee berry borer (CBB), Hypothenemus hampei Ferrari (Coleoptera: Curculionidae: Scolytinae), is one of the major insect pests of coffee worldwide. The present study was designed to assess the level of infestation of coffee berries at different developmental stages across different altitudes and coffee management systems. The experiment was carried out at three locations in southwestern Ethiopia under two coffee management systems and four coffee berry development stages with three replications. Results of the study showed significantly highest proportion of damaged berries (37.5%), number of holes per berry (10.88) and number of adult CBB per berry (7.55) on dried leftover berries at low-altitude study sites. On the other hand, the lowest mean percent damaged berries, number of holes per berry and number of adults were recorded at mid- and high-altitude study sites. The study also showed that, CBB caused significantly highest damage in plantation coffee management system than garden coffee. Results of this study highlight proper harvesting at red ripe stage in order to minimise incidence of CBB. It is also important to design integrated management strategies to mitigate CBB damage especially in lowland plantation coffee production systems.