The occurrence of Melissococcus plutonius in healthy colonies of Apis mellifera and the efficacy of European foulbrood control measures

European foulbrood (EFB) persists in England and Wales despite current treatment methods, all of which include feeding honey bee colonies with the antibiotic oxytetracycline (OTC). A large-scale field experiment was conducted to monitor a husbandry-based method, using comb replacement (known as Shook swarm), as a drug free EFB control option. The understanding of EFB epidemiology is limited, with little information on the presence of Melissococcus plutonius in disease free colonies. Additional samples were collected from diseased and disease free apiaries to identify symptomless infection. EFB reoccurrence was not significantly different between OTC and husbandry methods and real-time PCR data demonstrated that fewer Shook swarm treated colonies contained M. plutonius carryover to the Spring following treatment. Asymptomatic colonies from diseased apiaries showed an increased risk of testing positive for M. plutonius compared to asymptomatic colonies from disease free apiaries. The probability of a sample being symptomatic increased when a greater quantity of M. plutonius was detected in adult bees and larvae. The possibility of treating EFB as an apiary disease rather than a colony disease and the implications of a control strategy without antibiotics are discussed.     

Eastern filbert blight disease resistance from <Emphasis Type="Italic">Corylus americana</Emphasis> ‘Rush’ and selection ‘Yoder #5’ maps to linkage group 7

Eastern filbert blight (EFB), caused by the pyrenomycete Anisogramma anomala, is a serious threat to the hazelnut industry in the Pacific Northwest. EFB is endemic in eastern North America where it occasionally produces small cankers on the wild American hazelnut (Corylus americana). In contrast, most cultivars of European hazelnut (Corylus avellana) are susceptible. Genetic resistance is the most promising disease control method and is an objective of the Oregon State University hazelnut breeding program. ‘Gasaway’ resistance, which is governed by a dominant allele at a single locus, has been extensively used in the program. However, ‘Gasaway’ and some of its offspring have been infected by EFB isolates from New Jersey, Minnesota, and Michigan. Efforts to create new cultivars with durable EFB resistance include identifying and studying new resistance sources. In this study, resistant accessions C. americana ‘Rush’ and interspecific hybrid selection ‘Yoder #5’ were crossed with susceptible C. avellana selections and the resulting segregating seedling populations were inoculated by either exposure of potted trees under a structure topped with diseased branches or field exposure supplemented by tying diseased branches to each tree. Disease response was scored when cankers were visible 20 months after inoculation. Resistance from both sources segregated in a 1:1 ratio, indicating control by a single locus and a dominant allele for resistance. DNA extracted from the seedlings was amplified with previously mapped microsatellite markers. Resistance from both C. americana ‘Rush’ and ‘Yoder #5’ was placed on linkage group 7 in the same position as resistance from C. avellana ‘Ratoli.’ Linked microsatellite markers B753, GB372, and B509 will be useful for marker-assisted selection and the pyramiding of genes for durable EFB resistance. Assessing response to EFB is challenging, whether the plants are inoculated under a structure topped with diseased wood or in a humidity chamber in the greenhouse, or by exposure in the field. The pathogen has a 2-year life cycle, and there is a 15-month wait between inoculation and symptom expression. A small number of escapes is commonly encountered, and resistant plants occasionally develop small cankers. Our approach of studying segregation ratios and then mapping with microsatellite markers should be a useful approach for disease resistance studies in many tree crops.     

Molecular epidemiology and population structure of the honey bee brood pathogen Melissococcus plutonius

Melissococcus plutonius is the causative agent of European foulbrood (EFB), which is a serious brood disease of the European honey bee (Apis mellifera). EFB remains a threat because of a poor understanding of disease epidemiology. We used a recently published multi-locus sequence typing method to characterise 206 M. plutonius isolates recovered from outbreaks in England and Wales over the course of 2 years. We detected 15 different sequence types (STs), which were resolved by eBURST and phylogenetic analysis into three clonal complexes (CCs) 3, 12 and 13. Single and double locus variants within CC3 were the most abundant and widespread genotypes, accounting for 85% of the cases. In contrast, CCs 12 and 13 were rarer and predominantly found in geographical regions of high sampling intensity, consistent with a more recent introduction and localised spread. K-function analysis and interpoint distance tests revealed significant geographical clustering in five common STs, but pointed to different dispersal patterns between STs. We noted that CCs appeared to vary in pathogenicity and that infection caused by the more pathogenic variants is more likely to lead to honey bee colony destruction, as opposed to treatment. The importance of these findings for improving our understanding of disease aetiology and control are discussed.     

De novo sequencing of hazelnut bacterial artificial chromosomes (BACs) using multiplex Illumina sequencing and targeted marker development for eastern filbert blight resistance

Bacterial artificial chromosome (BAC) libraries are widely used in map-based cloning of plant genes. Eastern filbert blight (EFB), caused by the pyrenomycete Anisogramma anomala (Peck) E. Müller, is a devastating disease of European hazelnut (Corylus avellana L.) in the Pacific Northwest. A dominant allele at a single locus from the obsolete pollenizer “Gasaway” confers complete resistance. Our map-based cloning efforts use a BAC library for “Jefferson” hazelnut, which is heterozygous for resistance. Screening the library with primer pairs designed from RAPD markers closely linked to the EFB resistance locus identified 38 BACs. We sequenced 28 of these BACs using Illumina technology, by multiplexing with barcoded adapters. De novo sequence assembly using the programs Velvet and SOPRA and further alignment using CodonCode Aligner generated contigs whose length ranged from 393 to 108,194 bp. The number of contigs per BAC ranged from 1 to 19, and estimated coverage of assembled BACs ranged from 64 % to 100 %. Preliminary analysis of the sequences identified 779 simple sequence repeats (SSRs), from which we developed 23 markers. Of these, 17 were assigned to linkage group 6 adjacent to the disease resistance locus, five were placed on other linkage groups, and one could not be assigned to a linkage group. The BAC sequences and new SSR markers will be useful for our efforts at map-based cloning of the disease resistance gene.     

Differential resistance across paternal genotypes of honey bee brood to the pathogenic bacterium Melissococcus plutonius

Melissococcus plutonius is a pathogenic bacterium affecting immature stages of the western honey bee (Apis mellifera) and leads to European foulbrood (EFB) disease. Despite EFB outbreaks increasing in frequency in several countries in recent decades, there is little knowledge on the epidemiology of M. plutonius or on the defence mechanisms of honey bees against this pathogen. Mating of honey bee queens with multiple males (polyandry) can be such a mechanism, as it has been shown to be beneficial to colony health and fitness. It is hypothesized that a high level of polyandry was selected for in response to pathogen pressure to maximize the probability that at least some patrilines among nestmates in a colony possess a high degree of resistance to specific pathogens, ultimately protecting colonies against infections. We show that M. plutonius infection provokes differential mortality among patrilines of immature honey bee workers. Such differences indicate a genetic origin of resistance against this pathogen—supporting the polyandry hypothesis—and open up avenues to improve control of EFB disease via selective breeding.     

Distribution of Melissococcus plutonius in Honeybee Colonies with and without Symptoms of European Foulbrood

A sensitive hemi-nested polymerase chain reaction (PCR) was used for detection of Melissococcus plutonius, the causative agent of European foulbrood (EFB). Sampling was made in Switzerland, where EFB is a widespread disease and incidences have increased in recent years. Larvae from brood samples with and without clinical signs of disease (n = 92) and honey (n = 92) from the same colonies were investigated. Individual larvae (n = 60) and pupae (n = 30) from diseased brood in single colonies were also investigated to study the distribution of the bacterium within the brood between larvae. M. plutonius was detected in larvae in all apiaries where symptoms of EFB could be seen, but not in all colonies judged as cases of EFB in the field, when healthy-looking larvae from such colonies were tested. The occurrence of the bacterium within the brood was not limited to larvae with symptoms only, but was mainly found in diseased larvae. The bacterium was also found in pupae. Healthy-looking larvae—even from heavily diseased combs—failed, in a number of cases, to amplify product in the PCR. M. plutonius could only be detected in 35% of the brood nest honey from clinically diseased colonies.     

Survey and molecular detection of Melissococcus plutonius,the causative agent of European Foulbrood in honeybees in Saudi Arabia

A large-scale field survey was conducted to screen major Saudi Arabian beekeeping locations for infection by Melissococcus plutonius. M. plutonius is one of the major bacterial pathogens of honeybee broods and is the causative agent of European Foulbrood disease (EFB). Larvae from samples suspected of infection were collected from different apiaries and homogenized in phosphate buffered saline (PBS). Bacteria were isolated on MYPGP agar medium. Two bacterial isolates, ksuMP7 and ksuMP9 (16S rRNA GenBank accession numbers, KX417565 and KX417566, respectively), were subjected to molecular identification using M. plutonius -specific primers, a BLAST sequence analysis revealed that the two isolates were M. plutonius with more than 98% sequence identity. The molecular detection of M. plutonius from honeybee is the first recorded incidence of this pathogen in Saudi Arabia. This study emphasizes the need for official authorities to take immediate steps toward treating and limiting the spread of this disease throughout the country.     

Antimicrobial activity of tea tree oil nanoparticles against American and European foulbrood diseases agents

Paenibacillus larvae and Melissococcus plutonius are the primary bacterial pathogens of honeybees and the causative agents of American and European foulbrood disease (AFB and EFB) respectively. Such diseases have been gaining importance since there are few therapeutic options beyond the reporting of microorganisms resistant to conventional antibiotics. Due to the inefficiency and/or low efficacy of some antibiotics, researches with nanotechnology represent, possibly, new therapeutic strategies. Nanostructured drugs have presented some advantages over the conventional medicines, such as slow, gradual and controlled release, increased bioavailability, and reduced side-effects, among others. In this study, in vitro antimicrobial activity of tea tree oil (TTO) nanoparticles against Paenibacillus species, including P. larvae and M. plutonius strains was evaluated. Minimal inhibitory concentration (MIC) in Mueller–Hinton or KSBHI broth by the microdilution method was assessed. TTO registered MIC values of 0.18–6.25%, while the MIC values obtained for the TTO nanoparticle were of 0.01–0.93%. The possible toxic effect of TTO and TTO nanoparticle has been assessed by the spraying application method in the concentrations higher than the MICs. Bee mortality was evident only in treatment with TTO and the TTO nanoparticles show no toxic effects after 7 days of observation. Our results showed for the first time that TTO nanoencapsulation presented a high activity against Paenibacillus species and M. plutonius strains showing that the use of nanotechnology may represent one alternative way for the treatment or prevention of AFB and EFB.     

Whole slurry fermentation of maleic acid-pretreated oil palm empty fruit bunches for ethanol production not necessitating a detoxification process

The yield of ethanol from oil palm empty fruit bunches (EFB) was increased on exploiting maleic acid pretreatment combined with fermentation of the pretreated whole slurry. The optimized conditions for pretreatment were to expose EFB to a high temperature (190 °C) with 1 % (w/v) maleic acid for a short time duration (3 min ramping to the set temperature with no holding) in a microwave digester. An enzymatic digestibility of 60.9 % (based on theoretical glucose yield) was exhibited using pretreated and washed EFB after 48 h of hydrolysis. Simultaneous saccharification and fermentation (SSF) of the whole slurry of pretreated EFB for 48 h resulted in 61.3 % theoretical yield of ethanol based on the initial amount of glucan in untreated EFB. These results indicate that maleic acid is a suitable catalyst not requiring detoxification steps for whole slurry fermentation of EFB for ethanol production, thus improving the process economics. Also, the whole slurry fermentation can significantly increase the biomass utilization by converting sugar from both solid and liquid phases of the pretreated slurry.     

Production of humic acids from oil palm empty fruit bunch by submerged fermentation with Trichoderma viride: Cellulosic substrates and nitrogen sources

The novelty of this study was to produce humic acids by submerged fermentation of empty fruit bunch (EFB) with Trichoderma viride and to investigate the effects of the cellulosic substrates and the organic sources of nitrogen on the biotechnological production of these acids. The results obtained indicate the potential application of EFB, a waste of oil palm processing, for humic acids production. Because EFB contains cellulose, hemicellulose and lignin, fermentations were also performed using these polymers as carbon sources, separately or in combination. After 120 h of fermentation, significant production of humic acids was observed only in cultures containing either EFB or a mixture of the three polymers. Use of either potato peptone or yeast extract as a nitrogen source yielded nearly identical patterns of fungal growth and production of humic acids. The data obtained from microscopic imaging of T. viride growth and sporulation in EFB, coupled with the determined rates of production of humic acids indicated that the production of these acids is related to T. viride sporulation. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:631–637, 2013     

Characterization of ethanol fermentation waste and its application to lactic acid production by Lactobacillus paracasei

In this study, an ethanol fermentation waste (EFW) was characterized for use as an alternative to yeast extract for bulk fermentation processes. EFW generated from a commercial plant in which ethanol is produced from cassava/rice/wheat/barley starch mixtures using Saccharomyces cerevisiae was used for lactic acid production by Lactobacillus paracasei. The effects of temperature, pH, and duration on the autolysis of an ethanol fermentation broth (EFB) were also investigated. The distilled EFW (DEFW) contained significant amounts of soluble proteins (2.91 g/l), nitrogen (0.47 g/l), and amino acids (24.1 mg/l). The autolysis of the EFB under optimum conditions released twice as much amino acids than in the DEFW. Batch fermentation in the DEFW increased the final lactic acid concentration, overall lactic acid productivity, and lactic acid yield on glucose by 17, 41, and 14 %, respectively, in comparison with those from comparable fermentation in a lactobacillus growth medium (LGM) that contained 2 g/l yeast extract. Furthermore, the overall lactic acid productivity in the autolyzed then distilled EFW (ADEFW) was 80 and 27 % higher than in the LGM and DEFW, respectively.     

Bioconversion of lignocellulosic palm byproducts into enzymes and lipid by newly isolated oleaginous fungi

Direct conversion of palm pressed fiber (PPF) and palm empty fruit bunches (EFB) into enzymes and lipid by oleaginous fungi were performed through solid-state fermentation (SSF). Among the strains tested, TSIP9 converted PPF and EFB into lipid with the highest yield of 31.1 ± 1.7 mg/gram dry substrate (gds) and 37.5 ± 2.2 mg/gds, respectively. It also produced high activity of cellulolytic enzymes. It was identified as Aspergillus tubingensis. The similar fatty acids of its lipid to those of plant oil indicate its suitable use as biodiesel feedstock. The cellulase and xylanase production by this strain was improved when EFB was pretreated with alkaline. When alkaline-pretreated EFB was added with palm kernel cake (PK) as an alternative nitrogen source and the culture conditions were optimized through response surface methodology (RSM), the production of lipid, cellulase and xylanase were increased up to 88.5 ± 4.9 mg/gds, 26.1 ± 0.1 U/gds and 59.3 ± 0.3 U/gds, respectively. This study reveals the potential use of in situ cellulolytic enzymes producing fungi and the optimal conditions for direct conversion of lignocellulosic biomass into lipid.     

Conversion of acid hydrolysate of oil palm empty fruit bunch to L-lactic acid by newly isolated Bacillus coagulans JI12

Cost-effective conversion of lignocellulose hydrolysate to optically pure lactic acid is commercially attractive but very challenging. Bacillus coagulans JI12 was isolated from natural environment and used to produce L-lactic acid (optical purity?>?99.5 %) from lignocellulose sugars and acid hydrolysate of oil palm empty fruit bunch (EFB) at 50 °C and pH 6.0 without sterilization of the medium. In fed-batch fermentation with 85 g/L initial xylose and 55 g/L xylose added after 7.5 h, 137.5 g/L lactic acid was produced with a yield of 98 % and a productivity of 4.4 g/L?h. In batch fermentation of a sugar mixture containing 8.5 % xylose, 1 % glucose, and 1 % L-arabinose, the lactic acid yield and productivity reached 98 % and 4.8 g/L?h, respectively. When EFB hydrolysate was used, 59.2 g/L of lactic acid was produced within 9.5 h at a yield of 97 % and a productivity of 6.2 g/L?h, which are the highest among those ever reported from lignocellulose hydrolysates. These results indicate that B. coagulans JI12 is a promising strain for industrial production of L-lactic acid from lignocellulose hydrolysate.     

Nutritional evaluation of extruded faba bean (Vicia faba L.) as a protein supplement in cereals based diet in rats

The quality of extruded faba bean (EFB) (Vicia faba L.) as a protein supplement in cereals diet was evaluated. Growth rate and protein efficiency ratio was better in 20% EFB blended dietary group of rats than those fed either on cereals alone or at 40% EFB supplementation. Subsequent studies on digestibility co-efficient, biological value, net protein utilization and net protein ratio also confirmed biological utilization of EFB proteins in a better way at low level (20%).     

Production of lipids containing high levels of docosahexaenoic acid from empty palm fruit bunches by Aurantiochytrium sp. KRS101

The oleaginous microalga Aurantiochytrium sp. KRS101 was cultivated in enzymatic hydrolysates of alkali-pretreated empty palm fruit bunches (EFBs), without prior detoxification process. The maximal levels of lipid and docosahexaenoic acid synthesized were 12.5 and 5.4 g L^?1 after cultivation for 36 h. Similar lipid levels were also obtained via simultaneous saccharification and cultivation. The results suggested that EFB is a promising source for production of useful lipids by the microalgal strain.     

Decomposition and nutrient release temporal pattern of oil palm residues

The decomposition and nutrient release temporal patterns of three oil palm residues used as soil mulch were studied. Empty fruit bunches (EFB; 1000 kg plot^?1), Eco‐mat (processed EFB carpet; 30 kg plot^?1), and pruned palm fronds (180 kg plot^?1) were left to decompose (and sampled monthly) on the soil surface for 8 months. The frond's leaflets had the highest initial concentration for most nutrients, and the frond's rachis and Eco‐mat the lowest. The order of residue quality and rate of residue mass loss were: leaflets > fronds > EFB > Eco‐mat > rachis. EFB however had a higher mass loss rate than the fronds. Residue mass loss and nutrient release rates were faster at the beginning than at the end of the decomposition period. Leaflets released the highest total amount of nutrients (except for K), and rachis the lowest. The fronds released either significantly higher (for N and Ca) or not significantly different (for P and Mg) total amount of nutrients than EFB. Converting EFB into Eco‐mat had resulted in nutrient losses (e.g. N, K and Mg) and a residue quality reduction in Eco‐mat. This study's results would aid in better soil and oil palm fertilisation management.     

Diversity of Melissococcus plutonius from honeybee larvae in Japan and experimental reproduction of European foulbrood with cultured atypical isolates

European foulbrood (EFB) is an important infectious disease of honeybee larvae, but its pathogenic mechanisms are still poorly understood. The causative agent, Melissococcus plutonius, is a fastidious organism, and microaerophilic to anaerobic conditions and the addition of potassium phosphate to culture media are required for growth. Although M. plutonius is believed to be remarkably homologous, in addition to M. plutonius isolates with typical cultural characteristics, M. plutonius-like organisms, with characteristics seemingly different from those of typical M. plutonius, have often been isolated from diseased larvae with clinical signs of EFB in Japan. Cultural and biochemical characterization of 14 M. plutonius and 19 M. plutonius-like strain/isolates revealed that, unlike typical M. plutonius strain/isolates, M. plutonius-like isolates were not fastidious, and the addition of potassium phosphate was not required for normal growth. Moreover, only M. plutonius-like isolates, but not typical M. plutonius strain/isolates, grew anaerobically on sodium phosphate-supplemented medium and aerobically on some potassium salt-supplemented media, were positive for β-glucosidase activity, hydrolyzed esculin, and produced acid from L-arabinose, D-cellobiose, and salicin. Despite the phenotypic differences, 16S rRNA gene sequence analysis and DNA-DNA hybridization demonstrated that M. plutonius-like organisms were taxonomically identical to M. plutonius. However, by pulsed-field gel electrophoresis analysis, these typical and atypical (M. plutonius-like) isolates were separately grouped into two genetically distinct clusters. Although M. plutonius is known to lose virulence quickly when cultured artificially, experimental infection of representative isolates showed that atypical M. plutonius maintained the ability to cause EFB in honeybee larvae even after cultured in vitro in laboratory media. Because the rapid decrease of virulence in cultured M. plutonius was a major impediment to elucidation of the pathogenesis of EFB, atypical M. plutonius discovered in this study will be a breakthrough in EFB research.     

Parasitism,host feeding and immature development of Encarsia formosa reared from Trialeurodes vaporariorum and Bemisia tabaci on Trialeurodes ricini

Encarsia formosa Gahan (Hymenoptera: Aphelinidae), a thelytokous parasitoid, is an important biological control agent of whiteflies because of its outstanding reproduction and host‐feeding ability. In this study, we evaluated the parasitism, host feeding and developmental time of E. formosa populations reared on Trialeurodes vaporariorum (Westwood) (EFT) or on Bemisia tabaci (Gennadius) (EFB) when different nymphal instars of the castor whitefly, Trialeurodes ricini (Misra), were offered as hosts, with an aim to understand the preference of the parasitoid on nymphal instars of T. ricini. Experiments were conducted on castor bean plants at 26 ± 2°C, 50–60% RH and 16 : 8 (L : D) photoperiod. The results showed that E. formosa successfully oviposited and fed on all nymphal instars of T. ricini. However, numbers of the first instars fed on by the E. formosa populations reared on T. vaporariorum (EFT) and B. tabaci (EFB) were significantly greater (45.9 and 31.3, respectively) than those of the second (EFT: 30.4 and EFB: 15.8), the third (EFT: 22.4 and EFB: 13.2) and the fourth nymphal instars (EFT: 6.0 and EFB: 3.8). The number of T. ricini nymphs parasitized by E. formosa varied significantly among different instars, and the parasitism rates on the first instar (EFT: 15.2; EFB: 7.7) and fourth instar (EFT: 19.3; EFB: 4.9) were greater than those on the second and third instars. Encarsia formosa reared on T. vaporariorum had a significantly higher host feeding and ovipositing potential on T. ricini than EFB. When parasitizing the fourth instar nymphs, E. formosa completed development in a significantly shorter time (12.9 day) than when ovipositing in other instars (17.8–19.1 day). These results showed that EFT had a better host adaption than EFB. The information from this study should be useful for us to better understand the performance and nymphal preference of E. formosa from T. vaporariorum and B. tabaci when they parasitized and fed on T. ricini, and the interactions of parasitoids with different host whitefly species.     

Improvement of production of citric acid from oil palm empty fruit bunches: Optimization of media by statistical experimental designs

A sequential optimization based on statistical design and one-factor-at-a-time (OFAT) method was employed to optimize the media constituents for the improvement of citric acid production from oil palm empty fruit bunches (EFB) through solid state bioconversion using Aspergillus niger IBO-103MNB. The results obtained from the Plackett–Burman design indicated that the co-substrate (sucrose), stimulator (methanol) and minerals (Zn, Cu, Mn and Mg) were found to be the major factors for further optimization. Based on the OFAT method, the selected medium constituents and inoculum concentration were optimized by the central composite design (CCD) under the response surface methodology (RSM). The statistical analysis showed that the optimum media containing 6.4% (w/w) of sucrose, 9% (v/w) of minerals and 15.5% (v/w) of inoculum gave the maximum production of citric acid (337.94 g/kg of dry EFB). The analysis showed that sucrose (p < 0.0011) and mineral solution (p < 0.0061) were more significant compared to inoculum concentration (p < 0.0127) for the citric acid production.