<Emphasis Type="Italic">Metschnikowia</Emphasis> mating genomics

Genes involved in mating type determination and recognition were examined in Metschnikowia and related species, to gather insights on factors affecting mating compatibility patterns among haplontic, heterothallic yeast species of the genus. We confirmed the universality of the special mating locus organisation found in Clavispora lusitaniae across and exclusive to the family Metschnikowiaceae (i.e., Metschnikowia and Clavispora). Timing of the divergence between idiomorphs was confirmed to coincide with the origin of the larger (CUG-ser) clade comprising the Debaryomycetaceae and the Metschnikowiaceae, exclusive of Cephaloascus fragrans. The sequence of the a mating pheromone is highly conserved within the large-spored Metschnikowia species, including Metschnikowia orientalis and Metschnikowia hawaiiana, but not Metschnikowia drosophilae or Metschnikowia torresii, which have a pattern of their own, as do other clades in the genus. In contrast, variation in α pheromones shows a more continuous, although imperfect correlation with phylogenetic distance as well as with in vivo mating compatibility.     

Life History and Mating Behavior of <Emphasis Type="Italic">Rhagoletis solanophaga</Emphasis> (Diptera: Tephritidae), a Non-Diapausing Species with Highly Variable Mating Duration

As an initial contribution to understanding the adaptive value of behavioral and life-history strategies, the life cycle and mating behavior of an unstudied species of tephritid fruit fly in the genus Rhagoletis are characterized for the first time. Over a 9-month fruiting period, a small proportion of Solanum appendiculatum Dunal (< 10 %) was found to be infested with a single larva of Rhagoletis solanophaga (Hernández & Frías). The average duration of R. solanophaga lifecycle (c.a. 140 days from egg laying to death of adults) exceeded the three month fruitless period. Additionally, R. solanophaga is capable of exploiting Solanaceous plants in at least two genera. These features could have selected for a non-diapausing species of Rhagoletis, a genus where most species are univoltine. Nevertheless, some individuals in the population became dormant. As other members of the genus, R. solanophaga exhibited a resource defense mating system with forced copulations and multiple mating. Both males and females could be highly promiscuous and individual mating success exhibited a wide range of outcomes. Regardless of mating success, mated females stored similar amounts of sperm in two spherical spermathecae. Long copulations were observed, perhaps functioning as a form of mate guarding with probable disadvantages for females. We outline hypotheses and opportunities for future comparative studies examining sperm competition and mate guarding.     

A preliminary study on distributions and oviposition sites of <Emphasis Type="Italic">Drosophila suzukii</Emphasis> (Diptera: Drosophilidae) and its parasitoids on wild cherry tree in Tokyo,central Japan

Distributions and oviposition sites of Drosophila suzukii (Matsumura) and its parasitoids on wild cherry tree were studied in early summer in a suburb of Tokyo, central Japan. Adults of D. suzukii occurred in the foliage layer as well as in the undergrowth layer. The number of D. suzukii that emerged did not significantly differ between wild cherry fruit collected from the foliage layer and those from the undergrowth layer. In addition, the number of D. suzukii that emerged per fruit decreased when fruit were left on the ground longer. It is therefore assumed that D. suzukii females rarely oviposit eggs in fallen wild cherry fruit. The suzukii-associated type of Ganaspis brasiliensis (Ihering) was the major parasitoid that emerged from D. suzukii in the study area. The rate of parasitism by this parasitoid did not significantly differ between larvae in fresh fruit from the foliage layer and those in fallen fruit from the undergrowth layer. This may also suggest that this wasp rarely attacks D. suzukii larvae in fallen fruit. Adults of the suzukii-associated type of G. brasiliensis, Asobara sp. TK1, and Leptopilina japonica that attack D. suzukii were mainly collected from the foliage layer. On the basis of the present results, some proposals for the control of D. suzukii were discussed.     

Rearing system for the predatory phytoseiid <Emphasis Type="Italic">Euseius concordis</Emphasis> (Acari: Phytoseiidae)

Several species of predatory mites, especially those of the family Phytoseiidae, are potentially useful for the control of pest mites and insects. Among the phytoseiids, Euseius concordis (Chant), a species commonly found in South America, has been studied for possible use as biological control agent of pest mites. Given that Euseius species are known to feed on plant leaves and on pollen, the objective of this study was to determine plant species and pollen sources suitable to establish a pilot method of production of that species, based on a set of comparisons in the laboratory. Out of four plant species evaluated in the study, higher survivorship of E. concordis females in the absence of supplementary food was observed on leaflets of Canavalia ensiformis L. Out of two pollen types, higher survivorship was obtained on pollen of Typha domingensis L. In a subsequent step, the population of E. concordis increased 19.3× within 21 days when that pollen of T. domingensis was offered to the predator on plantlets of C. ensiformis, at 25.0 ± 1 °C, 70.0 ± 10% RH and 12 h photophase. Future studies may confirm the economic viability of this setup for the mass production of E. concordis.     

Molecular evidence for a natural diploid hybrid between <Emphasis Type="Italic">Miscanthus</Emphasis><Emphasis Type="Italic">sinensis</Emphasis> (Poaceae) and <Emphasis Type="Italic">M. sacchariflorus</Emphasis>

The hybrid origin of Miscanthus purpurascens has previously been proposed, primarily because of its intermediate morphology. In this study, phylogenies based on the DNA sequences from the internal transcribed spacer region of nuclear ribosomal DNA (nrDNA ITS), on the DNA sequences of the trnL intron and trnL-F intergenic spacer of chloroplast DNA, and on amplified fragment length polymorphism (AFLP) fingerprinting confirm that M. purpurascens originated through homoploid hybridization between M. sinensis and M. sacchariflorus. Two different types of ITS sequences were identified from almost all plants of M. purpurascens. One type was found to be closely related to M. sinensis and the other to M. sacchariflorus. Miscanthus purpurascens was found to possess many M. sinensis- and M. sacchariflorus-specific AFLP bands but no band specific to itself. Clustering with the Unweighted Pair Group Method with Arithmetic Mean and principal coordinate analysis based on the AFLP data also demonstrated that M. purpurascens is an approximate intermediate of the two species. In addition, M. purpurascens has the plastid genome of M. sinensis or M. sacchariflorus, suggesting that either species could be its maternal parent. All specimens of M. purpurascens and its coexisting parental species are identified as diploids (2n = 2x = 38). Possible mechanisms of natural hybridization, hybrid status, chloroplast DNA recombination, and evolutionary implications of this hybridization are also discussed.     

Mating Behavior and Basic Biology of <Emphasis Type="Italic">Haywardina cuculi</Emphasis><Emphasis Type="Bold">(</Emphasis>Diptera: Tephritidae), a Poorly Known Species Exhibiting High Variability in Copulation Duration

The natural history and mating behavior of a species of tephritid fruit fly in the poorly studied genus Haywardina are described for the first time. Haywardina cuculi Hendel larvae were recovered over four field seasons from infested fruit of Vassobia breviflora (Sendtn.) Hunz, which constitutes a new host plant record for this species. Recovered pupae emerged as adults over 183 days on average, suggesting that most individuals became dormant. Adult flies engaged in sexual activity as soon as two days after emergence, were highly promiscuous, and displayed large variability in copulation duration. As for most tephritid species in the subtribe Carpomyini, H. cuculi exhibited a resource defense mating system. Fly activity peaked around noon. Copulation could last from 9 min to 17 h, with most copulations beginning in the afternoon and lasting until the following day. We discuss the potential significance of copulation duration variability in light of mate guarding and sperm competition hypothesis and outline future research to understand the evolution of life history and these behavioral strategies.     

Expression of ethylene biosynthetic and signaling genes in relation to ripening of banana fruit after cold storage

Banana fruit are highly sensitive to chilling injury (CI), while the effect of different degrees of CI on the subsequent fruit ripening is largely unknown. In the present work, ripening characteristic of banana fruit after storage at 7 °C for 3 days or for 8 days, and expression levels of eight genes associated with ethylene biosynthetic and signaling, including MaACS1, MaACO1, MaERS1, MaERS3, and MaEIL1–4, were investigated. The results showed that banana fruit stored at 7 °C for 8 days exhibited more severe chilling symptoms than those at 7 °C for 3 days. Compared with banana fruit stored at 7 °C for 8 days, which showed abnormal ripening, more decrease in fruit firmness, while higher increase in ethylene production and hue angle were observed in banana fruit stored at 7 °C for 3 days, which could ripening normally. Moreover, gene expression profiles during ripening revealed that ethylene biosynthetic and signaling genes were differentially expressed in peel and pulp of banana fruit after storage at 7 °C for 3 days and 7 °C for 8 days. In the peel of fruit storage at 7 °C for 3 days, expression levels of MaACS1, MaACO1, MaEIL1, and MaEIL2 increased remarkably while MaERS3, MaEIL1, and MaEIL4 were enhanced in the fruit after storage at 7 °C for 8 days. In the pulp, with the exception of MaACO1 and MaERS3, expression levels of other genes did not exhibit a significant difference, between the banana fruit storage at 7 °C for 3 days and 7 °C for 8 days. Taken together, our results suggest that differential expression of ethylene biosynthetic and signaling genes such as MaERS3, MaACO1, and MaEIL2, may be related to ripening behavior of banana fruit with different degrees of CI after cold storage.     

<Emphasis Type="SmallCaps">d</Emphasis>-Xylose fermentation,xylitol production and xylanase activities by seven new species of <Emphasis Type="Italic">Sugiyamaella</Emphasis>

Sixteen yeast isolates identified as belonging to the genus Sugiyamaella were studied in relation to D-xylose fermentation, xylitol production, and xylanase activities. The yeasts were recovered from rotting wood and sugarcane bagasse samples in different Brazilian regions. Sequence analyses of the internal transcribed spacer (ITS) region and the D1/D2 domains of large subunit rRNA gene showed that these isolates belong to seven new species. The species are described here as Sugiyamaella ayubii f.a., sp. nov. (UFMG-CM-Y607^T = CBS 14108^T), Sugiyamaella bahiana f.a., sp. nov. (UFMG-CM-Y304^T = CBS 13474^T), Sugiyamaella bonitensis f.a., sp. nov. (UFMG-CM-Y608^T = CBS 14270^T), Sugiyamaella carassensis f.a., sp. nov. (UFMG-CM-Y606^T = CBS 14107^T), Sugiyamaella ligni f.a., sp. nov. (UFMG-CM-Y295^T = CBS 13482^T), Sugiyamaella valenteae f.a., sp. nov. (UFMG-CM-Y609^T = CBS 14109^T) and Sugiyamaella xylolytica f.a., sp. nov. (UFMG-CM-Y348^T = CBS 13493^T). Strains of the described species S. boreocaroliniensis, S. lignohabitans, S. novakii and S. xylanicola, isolated from rotting wood of Brazilian ecosystems, were also compared for traits relevant to xylose metabolism. S. valenteae sp. nov., S. xylolytica sp. nov., S. bahiana sp. nov., S. bonitensis sp. nov., S. boreocarolinensis, S. lignohabitans and S. xylanicola were able to ferment d-xylose to ethanol. Xylitol production was observed for all Sugiyamaella species studied, except for S. ayubii sp. nov. All species studied showed xylanolytic activity, with S. xylanicola, S. lignohabitans and S. valenteae sp. nov. having the highest values. Our results suggest these Sugiyamaella species have good potential for biotechnological applications.     

Production of <Emphasis Type="Italic">trans</Emphasis>-10,<Emphasis Type="Italic">cis</Emphasis>-12-conjugated linoleic acid using permeabilized whole-cell biocatalyst of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Objective

To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica.

Results

Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 °C, pH 7, 200 rpm with 1.5 g sodium acetate l^?1, 100 g wet cells l^?1, and 25 g LA l^?1. Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l^?1 after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l^?1.

Conclusion

Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.

     

Fine-scale spatial genetic structure in the frankincense tree <Emphasis Type="Italic">Boswellia papyrifera</Emphasis> (Del.) Hochst. and implications for conservation

The fine-scale genetic structure and how it varies between generations depends on the spatial scale of gene dispersal and other fundamental aspects of species’ biology, such as the mating system. Such knowledge is crucial for the design of genetic conservation strategies. This is particularly relevant for species that are increasingly fragmented such as Boswellia papyrifera. This species occurs in dry tropical forests from Ethiopia, Eritrea and Sudan and is an important source of frankincense, a highly valued aromatic resin obtained from the bark of the tree. This study assessed the genetic diversity and fine-scale spatial genetic structure (FSGS) of two cohorts (adults and seedlings) from two populations (Guba-Arenja and Kurmuk) in Western Ethiopia and inferred intra-population gene dispersal in the species, using microsatellite markers. The expected heterozygosity (H _E) was 0.664–0.724. The spatial analyses based on kinship coefficient (F _ij) revealed a significant positive genetic correlation up to a distance of 130 m. Spatial genetic structure was relatively weak (Sp = 0.002–0.014) indicating that gene dispersal is extensive within the populations. Based on the FSGS patterns found, we estimate indirectly gene dispersal distances of 103 and 124 m for the two populations studied. The high heterozygosity, the low fixation index and the low Sp values found in this study are consistent with outcrossing as the (predominant) mating system in B. papyrifera. We suggest that seed collection for ex situ conservation and reforestation programmes of B. papyrifera should use trees separated by distances of at least 100 m but preferably 150 m to limit genetic relatedness among seeds from different trees.     

Cloning of <Emphasis Type="Italic">phaCAB</Emphasis> genes from thermophilic <Emphasis Type="Italic">Caldimonas manganoxidans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> for poly(3-hydroxybutyrate) (PHB) production

PHB biosynthesis pathway, consisting of three open reading frames (ORFs) that encode for β-ketothiolase (phaA _Cma , 1179 bp), acetoacetyl-CoA reductase (phaB _Cma , 738 bp), and PHA synthase (phaC _Cma , 1694 bp), of Caldimonas manganoxidans was identified. The functions of PhaA, PhaB, and PhaC were demonstrated by successfully reconstructing PHB biosynthesis pathway of C. manganoxidans in Escherichia coli, where PHB production was confirmed by OD₆₀₀, gas chromatography, Nile blue stain, and transmission electron microscope (TEM). The protein sequence alignment of PHB synthases revealed that phaC _Cma shares at least 60% identity with those of class I PHB synthase. The effects of PhaA, PhaB, and PhaC expression levels on PHB production were investigated. While the overexpression of PhaB is found to be important in recombinant E. coli, performances of PHB production can be quantified as follows: PHB concentration of 16.8 ± 0.6 g/L, yield of 0.28 g/g glucose, content of 74%, productivity of 0.28 g/L/h, and Mw of 1.41 MDa.     

Cold tolerance and invasive potential of the redbay ambrosia beetle (<Emphasis Type="Italic">Xyleborus glabratus</Emphasis>) in the eastern United States

Native Lauraceae (e.g. sassafras, redbay) in the southeastern USA are being severely impacted by laurel wilt disease, which is caused by the pathogen Raffaelea lauricola T. C. Harr., Fraedrich and Aghayeva, and its symbiotic vector, the redbay ambrosia beetle (Xyleborus glabratus Eichhoff). Cold temperatures are currently the only viable limitation to the establishment of X. glabratus in northern populations of sassafras. The observed lower lethal temperature of X. glabratus (? 10.0 °C) is warmer than its supercooling point (? 22.0 °C), indicating the beetle is a freeze intolerant and chill susceptible species. Empirically derived X. glabratus lower lethal temperature thresholds were combined with host distribution and microhabitat-corrected climate data to produce species distribution models for X. glabratus in the eastern USA. Macroclimate data (30-year mean annual minimum temperature) were corrected (? 1.2 °C) to account for thermal buffering afforded to X. glabratus while living inside sassafras trees. Only 0.1% of the current US sassafras spatial extent experiences sufficiently harsh winters (locales where mean annual minimum winter temperatures ≤ ? 6.2 °C for ≥ 12 h) to exclude X. glabratus establishment in our species distribution model. Minimum winter temperatures will likely cause some X. glabratus mortality in ~ 52% of the current spatial extent of sassafras, although current data do not allow a quantification of X. glabratus mortality in this zone. Conversely, ~ 48% of the current spatial extent of sassafras is unlikely to experience sufficiently cold winter temperatures to cause any significant impediment to X. glabratus spread or establishment. A modest climate change scenario (RCP4.5) of + 1.4 °C would result in 91% of the current spatial extent of sassafras in the eastern USA occurring where winter minimum temperatures are unlikely to cause any mortality to X. glabratus.     

Genetic mapping reveals a candidate gene (<Emphasis Type="Italic">ClFS1</Emphasis>) for fruit shape in watermelon (<Emphasis Type="Italic">Citrullus lanatus</Emphasis> L.)

Key message

A 159 bp deletion in ClFS1 gene encoding IQD protein is responsible for fruit shape in watermelon.

Abstract

Watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] is known for its rich diversity in fruit size and shape. Fruit shape has been one of the major objectives of watermelon breeding. However, the candidate genes and the underlying genetic mechanism for such an important trait in watermelon are unknown. In this study, we identified a locus on chromosome 3 of watermelon genome controlling fruit shape. Segregation analysis in F₂ and BC₁ populations derived from a cross between two inbred lines “Duan125” (elongate fruit) and “Zhengzhouzigua” (spherical fruit) suggests that fruit shape of watermelon is controlled by a single locus and elongate fruit (OO) is incompletely dominant to spherical fruit (oo) with the heterozygote (Oo) being oval fruit. GWAS profiles among 315 accessions identified a major locus designated on watermelon chromosome 3, which was confirmed by BSA-seq mapping in the F₂ population. The candidate gene was mapped to a region 46 kb on chromosome 3. There were only four genes present in the corresponding region in the reference genome. Four candidate genes were sequenced in this region, revealing that the CDS of Cla011257 had a 159 bp deletion which resulted in the omission of 53 amino acids in elongate watermelon. An indel marker was derived from the 159 bp deletion to test the F₂ population and 105 watermelon accessions. The results showed that Cla011257 cosegregated with watermelon fruit shape. In addition, the Cla011257 expression was the highest at ovary formation stage. The predicted protein of the Cla011257 gene fitted in IQD protein family which was reported to have association with cell arrays and Ca²⁺-CaM signaling modules. Clear understanding of the genes facilitating the fruit shape along with marker association selection will be an effective way to develop new cultivars.

     

Enhanced isopropanol and <Emphasis Type="Italic">n</Emphasis>-butanol production by supplying exogenous acetic acid via co-culturing two clostridium strains from cassava bagasse hydrolysate

The focus of this study was to produce isopropanol and butanol (IB) from dilute sulfuric acid treated cassava bagasse hydrolysate (SACBH), and improve IB production by co-culturing Clostridium beijerinckii (C. beijerinckii) with Clostridium tyrobutyricum (C. tyrobutyricum) in an immobilized-cell fermentation system. Concentrated SACBH could be converted to solvents efficiently by immobilized pure culture of C. beijerinckii. Considerable solvent concentrations of 6.19 g/L isopropanol and 12.32 g/L butanol were obtained from batch fermentation, and the total solvent yield and volumetric productivity were 0.42 g/g and 0.30 g/L/h, respectively. Furthermore, the concentrations of isopropanol and butanol increased to 7.63 and 13.26 g/L, respectively, under the immobilized co-culture conditions when concentrated SACBH was used as the carbon source. The concentrations of isopropanol and butanol from the immobilized co-culture fermentation were, respectively, 42.62 and 25.45 % higher than the production resulting from pure culture fermentation. The total solvent yield and volumetric productivity increased to 0.51 g/g and 0.44 g/L/h when co-culture conditions were utilized. Our results indicated that SACBH could be used as an economically favorable carbon source or substrate for IB production using immobilized fermentation. Additionally, IB production could be significantly improved by co-culture immobilization, which provides extracellular acetic acid to C. beijerinckii from C. tyrobutyricum. This study provided a technically feasible and cost-efficient way for IB production using cassava bagasse, which may be suitable for industrial solvent production.     

Redirecting carbon flux through <Emphasis Type="Italic">pgi</Emphasis>-deficient and heterologous transhydrogenase toward efficient succinate production in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum is particularly known for its potentiality in succinate production. We engineered C. glutamicum for the production of succinate. To enhance C₃–C₄ carboxylation efficiency, chromosomal integration of the pyruvate carboxylase gene pyc resulted in strain NC-4. To increase intracellular NADH pools, the pntAB gene from Escherichia coli, encoding for transhydrogenase, was chromosomally integrated into NC-4, leading to strain NC-5. Furthermore, we deleted pgi gene in strain NC-5 to redirect carbon flux to the pentose phosphate pathway (PPP). To solve the drastic reduction of PTS-mediated glucose uptake, the ptsG gene from C. glutamicum, encoding for the glucose-specific transporter, was chromosomally integrated into pgi-deficient strain resulted in strain NC-6. In anaerobic batch fermentation, the production of succinate in pntAB-overexpressing strain NC-5 increased by 14% and a product yield of 1.22 mol/mol was obtained. In anaerobic fed-batch process, succinic acid concentration reached 856 mM by NC-6. The yields of succinate from glucose were 1.37 mol/mol accompanied by a very low level of by-products. Activating PPP and transhydrogenase in combination led to a succinate yield of 1.37 mol/mol, suggesting that they exhibited a synergistic effect for improving succinate yield.     

Simple Sequence Repeat and <Emphasis Type="Italic">S</Emphasis>-locus Genotyping to Explore Genetic Variability in Polyploid <Emphasis Type="Italic">Prunus spinosa</Emphasis> and <Emphasis Type="Italic">P. insititia</Emphasis>

Polyploid Prunus spinosa (2n = 4×) and P. insititia (2n = 6×) represent enormous genetic potential in Central Europe, which can be exploited in breeding programmes. In Hungary, 17 cultivar candidates were selected from wild-growing populations including 10 P. spinosa, 4 P. insititia and three P. spinosa × P. domestica hybrids (2n = 5×). Their taxonomic classification was based on their phenotypic characteristics. Six simple sequence repeats (SSRs) and the multiallelic S-locus genotyping were used to characterize genetic variability and reliable identification of the tested accessions. A total of 98 SSR alleles were identified, which presents 19.5 average allele number per locus, and each of the 17 genotypes could be discriminated based on unique SSR fingerprints. A total of 23 S-RNase alleles were identified. The complete and partial S-genotype was determined for 8 and 9 accessions, respectively. The identification of a cross-incompatible pair of cultivar candidates and several semi-compatible combinations help maximize fruit set in commercial orchards. Our results indicate that the S-allele pools of wild-growing P. spinosa and P. insititia are overlapping in Hungary. A phylogenetic and principal component analysis confirmed the high level of diversity and genetic differentiation present within the analysed genotypes and helped clarify doubtful taxonomic identities. Our data confirm that S-locus genotyping is suitable for diversity studies in polyploid Prunus species. The analysed accessions represent huge genetic potential that can be exploited in commercial cultivation.     

Development of a genetic sexing strain in <Emphasis Type="Italic">Bactrocera carambolae</Emphasis> (Diptera: Tephritidae) by introgression of sex sorting components from <Emphasis Type="Italic">B. dorsalis</Emphasis>, Salaya1 strain

Background

The carambola fruit fly, Bactrocera carambolae Drew & Hancock is a high profile key pest that is widely distributed in the southwestern ASEAN region. In addition, it has trans-continentally invaded Suriname, where it has been expanding east and southward since 1975. This fruit fly belongs to Bactrocera dorsalis species complex. The development and application of a genetic sexing strain (Salaya1) of B. dorsalis sensu stricto (s.s.) (Hendel) for the sterile insect technique (SIT) has improved the fruit fly control. However, matings between B. dorsalis s.s. and B. carambolae are incompatible, which hinder the application of the Salaya1 strain to control the carambola fruit fly. To solve this problem, we introduced genetic sexing components from the Salaya1 strain into the B. carambolae genome by interspecific hybridization.

Results

Morphological characteristics, mating competitiveness, male pheromone profiles, and genetic relationships revealed consistencies that helped to distinguish Salaya1 and B. carambolae strains. A Y-autosome translocation linking the dominant wild-type allele of white pupae gene and a free autosome carrying a recessive white pupae homologue from the Salaya1 strain were introgressed into the gene pool of B. carambolae. A panel of Y-pseudo-linked microsatellite loci of the Salaya1 strain served as markers for the introgression experiments. This resulted in a newly derived genetic sexing strain called Salaya5, with morphological characteristics corresponding to B. carambolae. The rectal gland pheromone profile of Salaya5 males also contained a distinctive component of B. carambolae. Microsatellite DNA analyses confirmed the close genetic relationships between the Salaya5 strain and wild B. carambolae populations. Further experiments showed that the sterile males of Salaya5 can compete with wild males for mating with wild females in field cage conditions.

Conclusions

Introgression of sex sorting components from the Salaya1 strain to a closely related B. carambolae strain generated a new genetic sexing strain, Salaya5. Morphology-based taxonomic characteristics, distinctive pheromone components, microsatellite DNA markers, genetic relationships, and mating competitiveness provided parental baseline data and validation tools for the new strain. The Salaya5 strain shows a close similarity with those features in the wild B. carambolae strain. In addition, mating competitiveness tests suggested that Salaya5 has a potential to be used in B. carambolae SIT programs based on male-only releases.

     

Selection of Potential Probiotic <Emphasis Type="Italic">Lactobacillus</Emphasis> with Inhibitory Activity Against <Emphasis Type="Italic">Salmonella</Emphasis> and Fecal Coliform Bacteria

Three hundred and sixty presumptive lactic acid bacteria (LAB) isolated from pregnant sows, newborn, suckling, and weaned piglets were preliminarily screened for anti-Salmonella activity. Fifty-eight isolates consisting of Lactobacillus reuteri (n = 32), Lactobacillus salivarius (n = 10), Lactobacillus mucosae (n = 8), Lactobacillus johnsonii (n = 5), and Lactobacillus crispatus (n = 3) were selected and further characterized for probiotic properties including production of antimicrobial substances, acid and bile tolerance, and cell adherence to Caco-2 cells. Eight isolates including Lact. johnsonii LJ202 and Lact. reuteri LR108 were identified as potential probiotics. LJ202 was selected for further use in co-culture studies of two-bacterial and multiple-bacterial species to examine its inhibitory activity against Salmonella enterica serovar Enteritidis DMST7106 (SE7106). Co-culture of LJ202 and SE7106 showed that LJ202 could completely inhibit the growth of SE7106 in 10 h of co-culture. In co-culture of multiple-bacterial species, culturable fecal bacteria from pig feces were used as representative of multiple-bacterial species. The study was performed to examine whether interactions among multiple-bacterial species would influence antagonistic activity of LJ202 against SE7106 and fecal coliform bacteria. Co-culture of SE7106 with different combinations of fecal bacteria and probiotic (LJ202 and LR108) or non-probiotic (Lact. mucosae LM303) strains revealed that the growth of SE7106 was completely inhibited either in the presence or in the absence of probiotic strains. Intriguingly, LJ202 exhibited notable inhibitory activity against fecal coliform bacteria while LR108 did not. Taken together, the results of co-culture studies suggested that LJ202 is a good probiotic candidate for further study its inhibitory effects against pathogen infections in pigs.     

Low-Input Fermentations of <Emphasis Type="Italic">Agave tequilana</Emphasis> Leaf Juice Generate High Returns on Ethanol Yields

During tequila production, up to 75 % w/w of the Agave plant is discarded when leaves are removed from the stem. The discarded leaves represent an extensive amount of unexploited biomass that was used here for bioethanol production in no-input fermentations, where no acid or enzymatic hydrolysis, supplementation of nutrients or standardization of carbohydrate content occur. Ethanol yield from Agave leaf juice is unaffected by sterilization but reduced if fermentation is reliant solely on endogenous microorganisms. Non-Saccharomyces yeasts, including Kluyveromyces marxianus and Candida akabanensis, proved to be more robust than standard Saccharomyces spp. and yielded up to 88 % of the theoretical maximum ethanol from leaf juice. Combining leaf and stem juice, as from a whole plant, was predicted to maximize yield at up to 19,439 L/ha of ethanol from mature plants.