[Show abstract][Hide abstract] ABSTRACT: Since several years it is known that strains of the entomopathogenic fungus Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Ascomycota: Hypocreales) are able to colonize plants as a true endophyte. However, so far no integrated bioprocess engineering approaches have been published where fermentation and formulation strategies are combined to optimize colonization of oilseed rape plant tissues. We therefore aimed at investigating whether and how blastospore (BS) formation can be shifted to resilient submerged conidiospores (SCS) by introducing osmotic stress in different growth phases.
When 50 g/L NaCl was added after 48 h to a culture of B. bassiana a yield of 1.4 ± 0.1x1010 SCS/g sucrose in shake flasks and 1.8 ± 0.1x1010 SCS/g sucrose in a stirred tank reactor were obtained. In a bioreactor, 24 h after the addition of NaCl, the formation of BS slowed down, the respiratory quotient decreased and a shift from BS to SCS set in.
Following these steps, different formulation strategies, namely encapsulation, film coating and liquid formulation were evaluated. B. bassiana grew out of beads as well as on commercial fungicide-coated seeds. Due to the complete suppression of fungal growth on non-sterile soil, the most suitable option was a foliar application. A liquid formulation consisting of 0.1 % Triton X-114, 1 % molasses, 1 % titanium dioxide and 106 spores/mL was applied on leaf tips. After 14 days, the endophyte was detected by PCR and microscopic analysis in the leaves.
Further research should focus on formation of SCS and protection of plants colonized by B. bassiana against herbivorous insects.
Biological Control 09/2015; 88:26-36. DOI:10.1016/j.biocontrol.2015.05.002 · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Several vectoring insects, such as aphids and white fly, harbour secondary bacterial endosymbionts. When an insect vectors a persistently transmitted plant virus, this virus should pass through the insect gut into the haemolymph. The secondary bacteria, however, may affect the movement, persistence and replication of the viruses, thereby influencing the vectoring efficacy or specificity of the insect in respect to these plant viruses. This issue, however, has not yet been fully investigated. Hence, the aim of this primary study is to investigate the effect of secondary bacterial endosymbionts of Sitobion avenae clones in respect of the vector specificity and transmission efficiency of barley yellow dwarf virus, as well as to highlight in general the role of secondary bacteria in virus transmission. The experiments were performed according to standard protocols using one virus strain, BYDV-PAV, and four S. avenae clones harbouring different bacterial and genetic profiles. We found that all tested clones were able to transmit the tested virus strain. Therefore, it can be concluded that the secondary bacterial endosymbionts may affect transmission efficiency of S. avenae but not their vectoring specificity. That said, the clones did not significantly differ in their efficiency of virus transmission. However, this study is initial evidence of the effect of secondary bacteria endosymbionts of aphids on virus transmission and further investigation is, therefore, still required.
[Show abstract][Hide abstract] ABSTRACT: Agricultural production is directly affected by projected increases in air temperature and changes in precipitation. A multi-model ensemble of regional climate change projections indicated shifts towards higher air temperatures and changing precipitation patterns during the summer and winter seasons up to the year 2100 for the region of Goettingen (Lower Saxony, Germany). A second major controlling factor of the agricultural production is the infestation level by pests. Based on long-term field surveys and meteorological observations, a calibration of an existing model describing the migration of the pest insect Ceutorhynchus napi was possible. To assess the impacts of climate on pests under projected changing environmental conditions, we combined the results of regional climate models with the phenological model to describe the crop invasion of this species. In order to reduce systematic differences between the output of the regional climate models and observational data sets, two different bias correction methods were applied: a linear correction for air temperature and a quantile mapping approach for precipitation. Only the results derived from the bias-corrected output of the regional climate models showed satisfying results. An earlier onset, as well as a prolongation of the possible time window for the immigration of Ceutorhynchus napi, was projected by the majority of the ensemble members.
International Journal of Biometeorology 02/2015; 59(11). DOI:10.1007/s00484-015-0966-1 · 3.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cashew, Anacardium occidentale L. (Sapindales: Anacardiaceae), is an important cash crop in Benin. However, its production is threatened by several biotic factors, especially insects. In Benin, very few studies have focused on insects and just listed species commonly found on cashew worldwide. The present investigation fills this gap by presenting an exhaustive inventory of insect species associated with this crop in the country. The survey was carried out from September 2009 to August 2010 in 22 cashew orchards (5 young and 17 mature) distributed over three major agroecological zones where cashew is most produced in the country. Insects were collected using chemical knock-down technique and visual observation followed by capture with sweep net. In addition, infested plant organs were sampled and incubated to collect emerging insects. In total, 262 insect species were recorded and identified. Among them, the wood borer Apate terebrans Pallas, the leafminer Eteoryctis gemoniella Stainton, and the mirid bugs Helopeltis schoutedeni Reuter., and Helopeltis anacardii Miller., appeared as the most important insect species attacking cashew in Benin. Beneficial insects encountered included some predators, parasitoids, and pollinators. Few vertebrate predators were also recorded on the trees. Differences in agroecological conditions or in field cleanliness did not affect the number of insect species encountered in the cashew orchards. The results of this study represent an important baseline data for the design and implementation of strategies for cashew protection in Benin.
[Show abstract][Hide abstract] ABSTRACT: The larvae of the invasive maize pest Diabrotica virgifera virgifera (Coleoptera; Chrysomelidae, western corn rootworm) hatch in the soil in spring and search for maize roots following CO2 gradients. CO2 is one cue that might be used as an attractant towards soil insecticides, a mechanism already shown in laboratory experiments. This study compared the efficacy of several combinations of in or between-row applications of different rates of CO2-emitting capsules and/or soil insecticides (here tefluthrin) aimed at preventing root damage by the pest larvae under field conditions. CO2 emission of the capsules in the soil lasted up to 28 days with a peak after 21 days coinciding with the first larval hatch. The pest density in the soil was not high enough to cause root damage above the economic threshold. Furthermore all tefluthrin applications, regardless of whether at full, half or quarter rates effectively prevented root damage; thus CO2 did not significantly further increase this efficacy. In-row applications of tefluthrin with or without CO2-emitting capsules prevented root damage to a much larger extent (59–77 % on the node injury scale) than the between-row applications of tefluthrin with or without capsules (17–31 %). In conclusion, further research on belowground orientation and movement of D. v. virgifera larvae, as well as tests with combinations of CO2-emitting capsules and lower rates of soil insecticide are needed to potentially develop attract and kill strategies as a management option against this maize pest.
Journal of Pest Science 06/2014; 87(2). DOI:10.1007/s10340-013-0551-5 · 2.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Western corn rootworm larvae use CO2 to locate maize roots. However, the importance of CO2 as a specific orientation cue close to maize roots is not unequivocally investigated. This study aimed at elucidating the effect of CO2 emitting capsules in combination with a soil insecticide (Tefluthrin = attract and kill) within the root system. We hypothesised that the capsules would result in an aggregation of the larvae at the soil insecticide, thus increasing its efficacy. A non-destructive observation device was used to study larval distribution and behaviour.
Spatial analysis of distance indices (SADIE) revealed an aggregation of the larvae around the capsules in an attract and kill treatment after 4 hours which was not found at the conventional treatment without the capsules. However, larval mortality did not differ in both treatments.
CO2 is a weak attractant for western corn rootworm larvae within the root system. Consequently, an attract and kill strategy based on a CO2 product will not contribute to a better control compared to conventional Tefluthrin applications. Host specific compounds, combined with a CO2 source, should be used to target more larvae, making attract and kill feasible as a management option against this pest.
Pest Management Science 04/2014; 70(4). DOI:10.1002/ps.3602 · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Western corn rootworm (Diabrotica virgifera virgifera LeConte) larvae use carbon dioxide (CO2) to locate the roots of their hosts. This study investigated whether an encapsulated CO2 source (CO2-emitting capsules) is able to outcompete CO2 gradients established by corn root respiration in the soil. Furthermore, the following two management options with the capsules were tested in semifield experiments (0.5- to 1-m2 greenhouse plots): the disruption of host location and an "attract-and-kill" strategy in which larvae were lured to a soil insecticide (Tefluthrin) between the corn rows. The attract-and-kill strategy was compared with an application of Tefluthrin in the corn rows (conventional treatment) at 33 and 18% of the standard field application rate. Application of the CO2-emitting capsules 30 cm from the plant base increased CO2 levels near the application point for up to 20 d with a peak at day 10. Both the disruption of host location and an attract-and-kill strategy caused a slight but nonsignificant reduction in larval densities. The disruption of host location caused a 17% reduction in larval densities, whereas an attract-and-kill strategy with Tefluthrin added at 33 and 18% of the standard application rate caused a 24 and 27% reduction in larval densities, respectively. As presently formulated, the CO2-emitting capsules, either with or without insecticide, do not provide adequate control of western corn rootworm.
[Show abstract][Hide abstract] ABSTRACT: The complex interactions among the maize pest Western Corn Rootworm (WCR), Glomus intraradices (GI-recently renamed Rhizophagus intraradices) and the microbial communities in both rhizosphere and endorhiza of maize have been investigated in view of new pest control strategies. In a greenhouse experiment, different maize treatments were established: C (control plants), W (plants inoculated with WCR), G (plants inoculated with GI), GW (plants inoculated with GI and WCR). After 20 days of WCR root feeding, larval fitness was measured. Dominant arbuscular mycorrhizal fungi (AMF) in soil and maize endorhiza were analyzed by cloning of 18S rRNA gene fragments of AMF, restriction fragment length polymorphism and sequencing. Bacterial and fungal communities in the rhizosphere and endorhiza were investigated by denaturing gradient gel electrophoresis of 16S rRNA gene and ITS fragments, PCR amplified from total community DNA, respectively. GI reduced significantly WCR larval development and affected the naturally occurring endorhiza AMF and bacteria. WCR root feeding influenced the endorhiza bacteria as well. GI can be used in integrated pest management programs, rendering WCR larvae more susceptible to predation by natural enemies. The mechanisms behind the interaction between GI and WCR remain unknown. However, our data suggested that GI might act indirectly via plant-mediated mechanisms influencing the endorhiza microbial communities.
Frontiers in Microbiology 12/2013; 4:357. DOI:10.3389/fmicb.2013.00357 · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Predicting western corn rootworm (Diabrotica virgifera virgifera LeConte; WCR) development in the field depends on models that use experimentally determined degree-days (°D). For constant temperature regimes, this temperature sum can be reliably used to predict hatch and development of WCR larvae. In the first experiment in climate cabinets, we evaluated the effects of varying day–night (4 and 6 °C difference) temperature regimes compared to equivalent constant temperature regimes on hatch, development, and recovery of WCR larvae from a non-diapausing population. Relative to constant diurnal temperatures, varying day–night temperatures resulted in earlier larval hatch and accelerated larval development (especially when day–night temperatures differed by 6 °C) due to direct temperature effects (i.e., the Kaufmann-effect) and to enhanced plant growth. For WCR eggs, the temperature sums needed for hatch in the field are overestimated when they are determined by models based on constant experimental temperatures. Recovery of larvae from soil was not affected by temperature, but was positively associated with plant height. In a second experiment we evaluated whether the found effect of varying temperature ranges on the acceleration of larval hatch is also influenced by the level of these varying temperatures. Initial hatch started earlier by varying diurnal temperatures only under a low-temperature regime (14 ± 4 °C). For herbivorous insects like WCR, plant growth effects induced by varying temperature regimes may result in increased variation in temperature-based developmental parameters. Consideration of these effects will improve models that predict WCR hatching patterns and will improve the development and timing of control strategies.
Journal of Pest Science 09/2013; 86(3). DOI:10.1007/s10340-013-0496-8 · 2.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Temperate grasses are infected with endophytic fungi of the genus Neotyphodium. These fungi asymptomatically live inside the plant’s tissue enhancing resistance of their hosts to herbivores and abiotic stresses. Perennial ryegrass (Lolium perenne), infected with Neotyphodium lolii can be detrimental to grazing livestock causing a neural disease. This study aimed at determining the impact of grassland management on fungal incidence. In a rural district of Central Germany 90 sites differing in land use intensity (extensive, intermediate and intensive) and grassland use (pastures, mown pastures, and meadows) were investigated for the presence of N. lolii. Infections were detected in 41% of the sites sampled, but overall infection rates were low (5.8 ± 9.2%). Neither the intensity of land use nor the type of grassland influenced endophytic incidences. In contrast, the geology of the sampled sites had a significant impact on the incidence of N. lolii. Grass tillers collected from limestone sites showed significantly higher infection rates than from other soils. We hypothesize that the low field capacity and high amount of potassium on limestone sites favored the survival of infected grasses under drought stress. Therefore, geology should be taken into account when considering management strategies for this endophytic fungus.
[Show abstract][Hide abstract] ABSTRACT: Cocoa agroforests can significantly support biodiversity, yet intensification of farming practices is degrading agroforestry habitats and compromising ecosystem services such as biological pest control. Effective conservation strategies depend on the type of relationship between agricultural matrix, biodiversity and ecosystem services, but to date the shape of this relationship is unknown. We linked shade index calculated from eight vegetation variables, with insect pests and beneficial insects (ants, wasps and spiders) in 20 cocoa agroforests differing in woody and herbaceous vegetation diversity. We measured herbivory and predatory rates, and quantified resulting increases in cocoa yield and net returns. We found that number of spider webs and wasp nests significantly decreased with increasing density of exotic shade tree species. Greater species richness of native shade tree species was associated with a higher number of wasp nests and spider webs while species richness of understory plants did not have a strong impact on these beneficial species. Species richness of ants, wasp nests and spider webs peaked at higher levels of plant species richness. The number of herbivore species (mirid bugs and cocoa pod borers) and the rate of herbivory on cocoa pods decreased with increasing shade index. Shade index was negatively related to yield, with yield significantly higher at shade and herb covers<50%. However, higher inputs in the cocoa farms do not necessarily result in a higher net return. In conclusion, our study shows the importance of a diverse shade canopy in reducing damage caused by cocoa pests. It also highlights the importance of conservation initiatives in tropical agroforestry landscapes.
PLoS ONE 06/2013; 8(3):e56115. DOI:10.1371/journal.pone.0056115 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cereal aphids (Homoptera: Aphididae) are a periodical threat to winter wheat (Triticum aestivum L.) production worldwide, and outbreaks have been mainly related to increasing inputs of mineral fertilizers. The hypothesis of lower aphid abundance under organic fertilizer treatments, however, has not been tested at the species-specific level. In a 4-year study, we examined cereal aphid populations and plant parameters (dry biomass and nitrogen content) in winter wheat fields under low (legume-based), intermediate (legume-based plus organic liquid manure), and high (mineral) nitrogen intensities; low and intermediate intensities are characteristic of organic fields, while high intensities are inherent in conventional farming systems. Aphid species differed markedly in their response to fertilizer treatment. Unexpectedly, legume-based organic fields were related to higher abundances of the ear-colonizing species Sitobion avenae (F.), whereas manure-fertilized organic fields and conventional fields had significantly higher abundances of Metopolophium dirhodum (Wlk.) and Rhopalosiphum padi (L.). Nitrogen concentration of winter wheat increased with fertilizer intensity, and total aphid abundance (all species) was positively correlated with nitrogen content of grains in manure-fertilized organic fields and conventional fields, but was not correlated in legume-based organic fields dominated by S. avenae (89–96 % of the aphid community). Collectively, we demonstrate strong bottom-up effects that result in an improved performance of S. avenae in low-input systems. As total aphid abundance (all species) simultaneously decreased in legume-based organic fields, crop managers risk to underestimate pest damage by ignoring the impact of a single species, S. avenae, which has the greatest potential to reduce crop yield.
[Show abstract][Hide abstract] ABSTRACT: Western corn rootworm larvae are serious soil dwelling maize pests, and use carbon dioxide (CO2) to locate maize roots. The efficacy of insecticides can be enhanced by a combination with an attractant used in host finding, known as attract and kill. This study tested the use of CO2 emitting capsules as an attractant in combination with the soil insecticide tefluthrin. An observation device was developed to study the temporal and spatial distribution changes of the larvae and to test whether these are influenced by the application of the capsules. Furthermore it was evaluated to what extent larvae are killed by the insecticide in combination with the capsules and whether this could be used for an attract and kill strategy to manage this pest.
The observation device enabled recovery of 20–40% of the inserted larvae. The spatial analysis of distance indices revealed a sequence of spatial and temporal distribution patterns of the larvae in the root system. This sequence of spatial distribution was disrupted by an application of the capsules around which the larvae started to aggregate. Up to 40% mortality of the larvae with attract and kill was observed and thus could be increased over the conventional application (11% mortality) at lower application rates of tefluthrin. In conclusion an attract and kill strategy might be valuable to target this soil dwelling pest. Experiments under field conditions are needed to explore its potential as a management option for the western corn rootworm. Moreover, a further development of the capsules with host specific cues is needed to increase the attractiveness and subsequent mortality of the larvae.
[Show abstract][Hide abstract] ABSTRACT: Aphids feed on plant phloem sap, rich in sugars but poor in essential amino acids. However, sugars cause osmotic regulation problems for aphids, which they overcome by hydrolysing the sugars in their gut and polymerising the hydrolysis products into oligosaccharides, excreted with honeydew. Aphids harbour primary bacterial endosymbionts, which supply them with essential amino acids necessary for survival. They also harbour secondary (facultative) endosymbionts (sfS), some of which have a positive impact on life history traits, although it is not yet known whether they also play a role in providing effective tolerance to differing levels of water soluble carbohydrates (WSCs). We investigated the relationship between WSC content of cocksfoot cultivars and performance of clones of the English grain aphid Sitobion avenae F. We evaluated how clone genotype and their sfS modulate performance on these different cultivars. We therefore examined the performance of genetically defined clones of S. avenae, collected from different host plants, harbouring different sfS. The performance was tested on 10 Dactylis glomerata L. cultivars with varying WSC content. D. glomerata is known as a wild host plant for S. avenae and is also commercially planted. We found that high WSCs levels are responsible for the resistance of D. glomerata cultivars to specific S. avenae clones. The minimum level of WSCs conferring resistance to D. glomerata cultivars was 1.7% dw. Cultivars with a WSC content of 2.2% or higher were resistant to S. avenae and did not allow reproduction. Our results further indicate that sfS modulate to some extend host plant cultivar adaptation in S. avenae. This is the first study revealing the importance of WSCs for aphid performance. Cocksfoot cultivars with a high content of WSCs might be therefore considered for aphid control or used for resistance breeding in this and other grass species, including cereals.
PLoS ONE 01/2013; 8(1):e54327. DOI:10.1371/journal.pone.0054327 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In southern Benin, the use of cover crops to improve and maintain soil fertility is on the increase. The
present study investigated the effect of two leguminous cover crops, Canavalia ensiformis (L.) DC and
Sesbania rostrata Brem. & Oberm., planted at different dates before maize (Zea mays (L.)), and cowpea
(Vigna unguiculata L.) planted as border rows on infestations of maize by the pyralid Mussidia nigrivenella
Ragonot and of other cob-boring lepidopteran pests. In both trials, M. nigrivenella densities at harvest
tended to be higher in the maize alone than the legume treatments, but the effect depended on the
timing of planting of the cover crop in relation to that of maize. There were no discernible trends for
other borers such as the noctuid Sesamia calamistis Hampson, the pyralid Eldana saccharina Walker, and
the tortricid Thaumatotibia leucotreta Meyrick. Furthermore, M. nigrivenella pest loads were considerably
higher on C. ensiformis than maize, indicating that the presence of alternative host plant species in the
vicinity of maize fields did not increase M. nigrivenella attack on maize. Though in some of the legume
treatments, grain damage and grain losses were higher than in the maize alone plots, per area yields did
not vary significantly.
[Show abstract][Hide abstract] ABSTRACT: Bacterial endosymbionts of Sitobion avenae: transmission and ecological interactions The primary symbiotic bacterium of aphids is critical for their growth and reproduction, while the secondary bacteria of aphids may interact with other life history traits like performance on host plants, and resistance to natural enemies. Transmission of secondary bacteria among different taxa in nature is still unclear. We studied the abundance of secondary bacteria in populations of Sitobion avenae and investigated the relationship between their abundance and the climatic conditions. We hypothesized that the symbiotic bacteria function differently under different climatic conditions. Vertical and horizontal transmission of the secondary bacteria can take place among and within aphid species and bacteria can also be transmitted to parasitoids. Furthermore, we were able to show that symbiotic bacteria enhance the tolerance to specific climatic condition and this contributes to the acclimation of parasitoids and aphids to different climatic conditions. Furthermore Also, we proved that bacteria-free phages are not responsible for aphid resistance against parasitoids, but in contrast the bacteria positively enhance fitness and hatching under certain climatic conditions. Finally, we investigated the role of secondary bacteria in host plant residence; we hypothesized that these bacteria could detoxify some chemicals which affect the performance of aphids on certain host plants. The ecological implications for interactions between secondary bacteria, aphids, and parasitoids, respectively, are discussed in this study.
Entomological Society of America Annual Meeting 2012; 11/2012
[Show abstract][Hide abstract] ABSTRACT: The predatory mite Typhlodromalus aripo and the entomopathogenic fungus Neozygites tanajoae, both introduced from Brazil for control of the cassava green mite (CGM) Mononychellus tanajoa, now co-occur in cassava fields in Benin. However, studies on interactions between these two natural enemies and how they might affect CGM biological control are lacking. We determined in screenhouse experiments the effects of single and combined releases of N. tanajoae and T. aripo on CGM suppression. In the single natural enemy treatment, both T. aripo and N. tanajoae significantly reduced CGM densities, but the results of the predator (T. aripo) are more quickly measurable than those of the pathogen (N. tanajoae) in our short-term experiment. The level of CGM suppression in the combined natural enemy treatment was reduced considerably compared with T. aripo-alone, but only slightly when compared with N. tanajoae alone, with a simultaneous reduction in T. aripo and N. tanajoae abundance or prevalence. In a laboratory experiment, T. aripo fed more on N. tanajoae-infected CGM than on healthy CGM and its oviposition and survival were reduced when fed on the former compared with the latter, which can help in explaining the reduction in numbers of T. aripo and consequently the considerable loss in suppression of CGM in the combined natural enemy treatment in the screenhouse experiment. Together, the screenhouse and the laboratory experiments predicted negative interactions between the two natural enemies with negative consequences for CGM biological control. Long-term field observations and rigorous field experiments that simultaneously manipulate T. aripo and N. tanajoae abundance and prevalence are needed to validate the prediction of this study.
[Show abstract][Hide abstract] ABSTRACT: The western corn rootworm (WCR) is one of the economically most important pests of maize. A better understanding of microbial communities associated with guts and eggs of the WCR is required in order to develop new pest control strategies, and to assess the potential role of the WCR in the dissemination of microorganisms, e.g., mycotoxin-producing fungi.
Total community (TC) DNA was extracted from maize rhizosphere, WCR eggs, and guts of larvae feeding on maize roots grown in three different soil types. Denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rRNA gene and ITS fragments, PCR-amplified from TC DNA, were used to investigate the fungal and bacterial communities, respectively. Microorganisms in the WCR gut were not influenced by the soil type. Dominant fungal populations in the gut were affiliated to Fusarium spp., while Wolbachia was the most abundant bacterial genus. Identical ribosomal sequences from gut and egg samples confirmed a transovarial transmission of Wolbachia sp. Betaproteobacterial DGGE indicated a stable association of Herbaspirillum sp. with the WCR gut. Dominant egg-associated microorganisms were the bacterium Wolbachia sp. and the fungus Mortierella gamsii.
The soil type-independent composition of the microbial communities in the WCR gut and the dominance of only a few microbial populations suggested either a highly selective environment in the gut lumen or a high abundance of intracellular microorganisms in the gut epithelium. The dominance of Fusarium species in the guts indicated WCR larvae as vectors of mycotoxin-producing fungi. The stable association of Herbaspirillum sp. with WCR gut systems and the absence of corresponding sequences in WCR eggs suggested that this bacterium was postnatally acquired from the environment. The present study provided new insights into the microbial communities associated with larval guts and eggs of the WCR. However, their biological role remains to be explored.
PLoS ONE 10/2012; 7(10):e44685. DOI:10.1371/journal.pone.0044685 · 3.23 Impact Factor