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More and more software projects are being consolidated into ecosystems to increase their discovery, usability, and usefulness. Some of the most popular ecosystems exist in npmjs, Python Package Indexing, and Apache Maven Repository. It is difficult for developers to relate these projects and use them to their full potential because of their number, the spread and depth of their features, and their intrinsic and accidental complexities. We present a technique—SECO Storms Maker—to capture and present the essential information from projects in an ecosystem to help developers join, use, and contribute to the ecosystem. We generate word-clouds based on the projects’ documentation via tokenization and distribution frequency. We identify relations among projects using grammar patterns scanning after part-of-speech tagging. We put these word-clouds into a graph to ease navigation and exploration. We evaluate our technique by manually building a ground truth and comparing a randomly-selected project with SECO to show its benefits.

Currently, the genetic modification of Aspergillus oryzae is mainly dependent on protoplastmediated transformation (PMT). In this study, we established a dual selection marker system in an industrial A. oryzae 3.042 strain by using Agrobacterium tumefaciens-mediated transformation (ATMT). We first constructed a uridine/uracil auxotrophic A. oryzae 3.042 strain and a pyrithiamine (PT)-resistance binary vector. Then, we established the ATMT system by using uridine/uracil auxotrophy and PT-resistance genes as selection markers. Finally, a dual selection marker ATMT system was developed. This study demonstrates a useful dual selection marker transformation system for genetic manipulations of A. oryzae 3.042.

Genetic engineering of the filamentous fungus Aspergillus oryzae still requires more suitable selection markers for fungal transformation. Our previous work has shown that Agrobacterium tumefaciens-mediated transformation (ATMT) based on the uridine/uracil auxotrophic mechanism with pyrG as the selection marker is very efficient for gene transfer in A. oryzae. In the present study, we delete the hisB gene, which is essential for histidine biosynthesis, in A. oryzae via homologous recombination and demonstrate that hisB is a reliable selection marker for genetic transformation of this fungus. Under optimal conditions, the ATMT efficiency of the histidine auxotrophic A. oryzae reached 515 transformants per 106 spores. Especially, we have succeeded in constructing a new ATMT system based on dual auxotrophic A. oryzae mutants with two different selection markers including hisB and pyrG. This dual auxotrophic ATMT system displayed a transformation efficiency of 232 transformants per 106 spores for the hisB marker and 318 transformants per 106 spores for the pyrG marker. By using these selectable markers, the co-expression of the DsRed and GFP fluorescent reporter genes was implemented in a single fungal strain. Furthermore, we could perform both the deletion and complementation of the laeA regulatory gene in the same strain of A. oryzae to examine its function. Conclusively, the ATMT system constructed in our work represents a promising genetic tool for studies on recombinant expression and gene function in the industrially important fungus A. oryzae.

REST APIs are nowadays the de-facto standard for Web applications. However, as more systems and services adopt the REST architectural style, many problems arise regularly. To avoid these repetitive problems, developers should follow good practices and avoid bad practices. Thus, research on good and bad practices and how to design a simple but effective REST API are essential. Yet, to the best of our knowledge, there are only a few concrete solutions to recurring REST API practices, like “API Versioning”. There are works on defining or detecting some practices, but not on solutions to the practices. We present the most up-to-date list of REST API practices and formalize them in the form of REST API (anti)patterns. We validate our design (anti)patterns with a survey and interviews of 55 developers.

Land consolidation is an effective solution for the hindrances in agricultural production and rural development caused by land fragmentation. In the Red River Delta of Vietnam, where land is still highly fragmented, the application of land consolidation is required. By using a bottom-up approach, the paper aims to clarify the effect of land consolidation on farm households in selected communities (as case studies) of two provinces (Hung Yen and Vinh Phuc) in the Red River Delta. With the primary structured and semi-structured interview method, 172 household questionnaires and 22 in-depth questionnaires (from local officials) were collected. The results indicated that land consolidation could either change the spatial structure or expand the area of land parcels, facilitate the conversion of crop structure, increase household incomes, accelerate mechanization in agricultural production, and create more job opportunities for agricultural laborers. However, we also found that the land consolidation process conducted in the case studies is inadequate and lacks integration with other related policies.

The electrical discharge drilling (EDD) process is an effective machining approach to produce various holes in difficult-to-cut materials. However, the energy efficiency of the EDD operation has not thoroughly been considered in published works. The aim of the current work is to optimize varied parameters for enhancing the material removal rate (MRR), saving drilled energy (ED), and decreasing the expansion of the hole (HE) for the EDD process. Three advanced factors, including the gap voltage adjustor (GAP), capacitance parallel connection (CAP), and servo sensitivity selection (SV), are considered. The predictive models of the performances were proposed with the support of the adaptive neuro-based fuzzy inference system (ANFIS). An integrative approach combining the analytic hierarchy process (AHP) and the neighborhood cultivation genetic algorithm (NCGA) was employed to select optimal factors. The findings revealed the optimal values of the CAP, GAP, and SV were 6, 5, and 4, respectively. Moreover, the ED and HE were decreased by 16.78% and 28.68%, while the MRR was enhanced by 89.72%, respectively, as compared to the common setting values. The explored outcome can be employed as a technical solution to enhance the energy efficiency, drilled quality, and productivity of the EDD operation.

Verticillium dahliae gehort zu den bodenburtigen Askomyceten. Dieser Pilz verursacht Welke-Erkrankungen und verfruhtes Altern in mehr als 200 verschiedenen, auch okonomisch wichtigen Pflanzen. Verticillium kann im Boden ohne Wirtspflanze durch die Bildung von Mikrosklerotien bis zu 10 Jahre uberleben. Wurzelexsudate induzieren die Auskeimung der Mikrosklerotien. V. dahliae infiziert seinen Wirt durch die Wurzel, besiedelt den Wurzelkortex und dringt dann in die Xylemgefase ein. Die Infektion des Wirts durch pathogene Pilze erfordert Penetrations- und Kolonisierungsprozesse. Am Eindringen durch die Wurzeloberflache sind adhasive Proteine an verschiedenen Stellen der Wirt-Parasit-Interaktion beteiligt. Adhasive Proteine sind in S. cerevisiae gut untersucht, wahrend nur wenig uber sie in V. dahliae bekannt ist. Der Adhasions-Transkriptionsfaktor Flo8 aus Hefe reguliert die Expression der sogenannten „Flocculation“-Gene wie zum Beispiel FLO1 und FLO11. Ein Stamm ohne FLO8 ist nicht in der Lage an Agarmedium zu haften und in Flussigmedium auszuflocken. Die im Zellkern lokalisierten Transkriptionsfaktoren Som1 und Vta3 konnen die Adhasion in einem S. cerevisiae Stamm, welchem FLO8 fehlt, wiederherstellen. Som1 und Vta3 induzieren die Expression von FLO1 und FLO11, welche Adhasine kodieren. Die SOM1 und VTA3 Gene wurden deletiert und ihre Funktion in der durch Pilze verursachten Pflanzenpathogenese wurde durch genetische, zellbiologische, Proteom- und Pflanzenpathogenitatsexperimente untersucht. V. dahliae Som1 und Vta3 sind sequenziell fur die Penetration und Kolonisation des Pflanzenwirts erforderlich. Som1 unterstutzt die pilzliche Adhasion sowie das Eindringen in die Wurzel. Somit wird es fruher fur die Besiedlung der Pflanzenwurzeloberflache und die Tomateninfektion benotigt als Vta3. Som1 kontrolliert daruber hinaus die Positionierung von Septen und die Grose von Vakuolen und folglich auch die Entwicklung von Hyphen inklusive der Bildung von Lufthyphen und normalen Hyphenverzweigungen. Som1 und Vta3 beeinflussen die Bildung von Konidien und Mikrosklerotien und wirken sich in der Antwort auf oxidativen Stress entgegen. Die molekulare Funktion von Som1 ist zwischen dem Pflanzenpathogen V. dahliae und dem opportunistischen Humanpathogen Aspergillus fumigatus konserviert. Som1 kontrolliert die Expression von Genen welche fur Adhasion und die Antwort auf oxidativen Stress benotigt werden. Sowohl Som1 als auch Vta3 regulieren ein genetisches Netzwerk fur die Bildung von Konidien und Mikrosklerotien sowie die Pathogenitat von V. dahliae.

Penicillium digitatum is a major postharvest pathogen of citrus crops. This fungus broadly spreads worldwide and causes green mold disease, which results in severe losses for citrus production. Understanding of the citrus infection by P. digitatum may help develop effective strategies for controlling this pathogen. In this study, we have characterized a virulent strain of P. digitatum isolated in Vietnam and established a highly efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for this fungal strain with two newly constructed binary vectors. These binary vectors harbor dominant selectable markers for hygromycin or nourseothricin resistance, and expression cassettes for the red fluorescent protein (DsRed) or the green fluorescent protein (GFP), respectively. Using the established ATMT system, the transformation efficiency of the Vietnamese strain could reach a very high yield of 1240±165 transformants per 106 spores. Interestingly, we found that GFP is much better than DsRed for in situ visualization of citrus fruit colonization by the fungus. Additionally, we showed that the transformation system can also be used to generate T-DNA insertion mutants for screening non-pathogenic or less virulent strains. Our work provides a new platform including a virulent tropical strain of P. digitatum, an optimized ATMT method and two newly constructed binary vectors for investigation of the postharvest pathogen. This platform will help develop strategies to dissect molecular mechanisms of host-pathogen interactions in more detail as well as to identify potential genes of pathogenicity by either insertional mutagenesis or gene disruption in this important pathogenic fungus.

Purpureocillium lilacinum (formerly Paecilomyces lilacinus) is widely commercialized for controlling plant-parasitic nematodes and represents a potential cell factory for enzyme production. This nematicidal fungus is intrinsically resistant to common antifungal agents used for genetic transformation. Therefore, molecular investigations in P. lilacinum are still limited so far. In the present study, we have established a new Agrobacterium tumefaciens-mediated transformation (ATMT) system in P. lilacinum based on the uridine/uracil auxotrophic mechanism. Here, uridine/uracil auxotrophic mutants were simply generated via UV irradiation instead of a complicated genetic approach for the pyrG gene deletion. A stable uridine/uracil auxotrophic mutant was then selected as a recipient for fungal transformation. We further indicated that the pyrG gene from Aspergillus niger can be used as a selectable marker for genetic transformation of P. lilacinum. Under optimized conditions for ATMT, the transformation efficiency reached 2873 ± 224 transformants per 106 spores. Using the constructed ATMT system, we succeeded in expressing the DsRed reporter gene in P. lilacinum. Additionally, we have identified a very promising mutant for chitinase production from a collection of T-DNA insertion transformants. This mutant possesses a special phenotype of hyper-branching mycelium and produces more conidia in comparison to the wild strain. Conclusively, our ATMT system can be exploited for overexpression of target genes or for T-DNA insertion mutagenesis in the agriculturally important fungus P. lilacinum. The genetic approach in the present work may also be applied for developing similar ATMT systems in other fungi, especially for fungi that their genome databases are currently not available.

The extraction of genomic DNA from microbial cells plays a significant role in PCR-based applications such as molecular diagnosis, microbial taxonomy, screening of genetically engineered microorganisms, and other such PCR-based applications. Currently, many methods for extraction of genomic DNA from microorganisms have been developed. However, these methods either require hazardous chemicals or consist of time-consuming steps for effective execution. In this study, we have established a simple and universal genomic DNA extraction method for different microorganisms including bacteria, yeasts, molds, and microalgae. Our method does not require harmful reagents such as phenol and chloroform for the extraction process to minimize the generation of hazardous wastes. The obtained genomic DNA products displayed high concentrations and represented a good purity level with the average 260 nm/280 nm absorbance ratios (A260/280) that range from 1.6 to 2.0. The DNA molecules further remained considerably intact when analyzed on agarose gels. More importantly, these DNA products were qualified through successful PCR amplifications of 16S rRNA gene, rDNA internal transcribed spacer (ITS), or 18S rRNA gene from genomes of bacteria, fungi, and microalgae respectively. Furthermore, with the extracted genomic DNA products, the processes of the identification of the haploid and diploid states of the Saccharomyces yeast strains or detection of putative strains of Aspergillus oryzae and Aspergillus flavus that have been isolated from infected food materials through PCR analyses are facilitated. The genomic DNA extraction method established in this study is easy to manage, time saving and cost-effective, and environmentally friendly.

Many fungi are able to produce resting structures, which ensure survival and protect them against various stresses in their habitat such as exposure to UV light, temperature variations, drought as well as changing pH and nutrient conditions. Verticillium dahliae is a plant pathogenic fungus that forms melanized resting structures, called microsclerotia, for survival of time periods without a host. These highly stress resistant microsclerotia persist in the soil for many years and are therefore problematic for an effective treatment of the fungus. The Verticillium transcription activator of adhesion 1 (Vta1) was initially identified as one of several transcriptional regulators that rescue adhesion in non-adhesive Saccharomyces cerevisiae cells. Vta2 and Vta3 are required for early steps in plant infection and colonization and additionally control microsclerotia formation. Here, we show that Vta1 function is different, because it is dispensable for root colonization and infection. Vta1 is produced in the fungal cell during microsclerotia development. Analysis of the deletion mutant revealed that the absence of Vta1 allows microsclerotia production, but they are colorless and no more melanized. Vta1 is required for melanin production and activates transcription of melanin biosynthesis genes including the polyketide synthase encoding PKS1 and the laccase LAC1. The primary function of Vta1 in melanin production is important for survival of microsclerotia as resting structures of V. dahliae.

Currently, the genetic modification of

Agricultural land conversion for urban development is a major process in the vicinity of many cities in Vietnam. This causes problems for the fabric of local society. In this paper, we evaluate four agricultural land’s acquisition projects in two areas (Hoai Duc district and Thanh Oai district) in Hanoi, Vietnam to examine two primary questions: (1) how do farmers obtain any economic benefit from urban development projects? And, (2) how are socio-economic livelihoods of the farmers effected by land conversion? The investigation relied on a market land price's survey using a structured-interview method, in which 395 land-acquired households were randomly selected. The responses indicate that there are unequal benefits among the various stakeholders. On average farmers tend to benefit the least. Although the farmers have the possibility to transform their agricultural livelihood into a non-agricultural one with probably a higher income, they are actually faced with many difficulties in maintaining non-agricultural activities, in finding stable alternative livelihood activities, and in using the compensation for investments.

Aspergillus oryzae is a filamentous fungus widely used in food industry and as a microbial cell factory for recombinant protein production. Due to the inherent resistance of A. oryzae to common antifungal compounds, genetic transformation of this mold usually requires auxotrophic mutants. In this study, we show that Agrobacterium tumefaciens-mediated transformation (ATMT) method is very efficient for deletion of the pyrG gene in different Aspergillus oryzae wild-type strains to generate uridine/uracil auxotrophic mutants. Our data indicated that all the obtained uridine/uracil auxotrophic transformants, which are 5- fluoroorotic acid (5-FOA) resistant, exist as the pyrG deletion mutants. Using these auxotrophic mutants and the pyrG selectable marker for genetic transformation via A. tumefaciens, we could get about 1060 transformants per 106 fungal spores. In addition, these A. oryzae mutants were also used successfully for expression of the DsRed fluorescent reporter gene under control of the A. oryzae amyB promoter by the ATMT method, which resulted in obvious red transformants on agar plates. Our work provides a new and effective approach for constructing the uridine/uracil auxotrophic mutants in the importantly industrial fungus A. oryzae. This strategy appears to be applicable to other filamentous fungi to develop similar genetic transformation systems based on auxotrophic/nutritional markers for food-grade recombinant applications.

The vascular plant pathogen Verticillium dahliae colonizes the xylem fluid where only low nutrient concentrations are provided. Biosynthesis of the vitamin thiamine is connected to oxidative stress. The highly conserved VdThi4 protein is localized in fungal mitochondria and is required under vitamin B1 limiting conditions. Deletion of the corresponding VdTHI4 gene by Agrobacterium-mediated transformation resulted in strains which were impaired in growth on thiamine-free medium and could be rescued by additional vitamin supply or by complementation with the original gene after protoplastation. Furthermore, we show that VdThi4 increases fungal stress tolerance such as UV-damage or oxidative stress. The orthologous sti35 gene of Fusarium oxysporum, another vascular wilt fungus, was shown to be involved in stress response, however to be dispensable for pathogenicity on tomato. In contrast, VdTHI4 is required for fungal-induced tomato disease demonstrated by infection assays with a V. dahliae ΔVdTHI4 deletion strain which is still able to invade plants through the roots but is asymptomatic. Our results suggest remarkable differences between two vascular tomato pathogens where VdThi4 is required for pathogenicity of V. dahliae, whereas F. oxysporum still causes disease when the corresponding Sti35 protein is absent.

Verticillium longisporum and Verticillium dahliae are soil borne vascular pathogens. Under standard laboratory growth conditions both fungi infect Arabidopsis thaliana ecotype Col-0 plants via primary and lateral (secondary) root tips and colonize the vascular cylinder including proto- and metaxylem. After entry into the hypocotyl both Verticillium species spread into petioles and leaves, but with different efficiency and distinct disease symptom patterns. V. longisporum typically induces early senescence whereas V. dahliae infections cause wilting. In both cases, these symptoms coincide with the switch from a biotrophic to a necrotrophic life style. Fungal proliferation analyses monitored by quantitative real-time PCR and anatomical studies show that V. dahliae is able to colonize aerial plant parts more efficiently compared to V. longisporum. Moreover, V. longisporum-infection triggers hyperplastic xylem formation and cellular transdifferentiation in Arabidopsis, whereas V. dahliae-infection induces enhanced xylem lignification. We have recently shown that V. longisporum induced de novo xylem formation results in enhanced drought stress tolerance, suggesting a conditionally mutualistic interaction. Here we show that on the contrary V. dahliae infections do not provide protection of the plant against drought. Together, our results demonstrate that V. dahliae and V. longisporum induce distinct and species-specific developmental and structural alterations in Arabidopsis, which differently affect plant fitness under concomitant abiotic drought stress conditions.

Summary Six transcription regulatory genes of the Verticillium plant pathogen, which reprogrammed nonadherent budding yeasts for adhesion, were isolated by a genetic screen to identify control elements for early plant infection. Verticillium transcription activator of adhesion Vta2 is highly conserved in filamentous fungi but not present in yeasts. The Magnaporthe grisea ortholog conidiation regulator Con7 controls the formation of appressoria which are absent in Verticillium species. Vta2 was analyzed by using genetics, cell biology, transcriptomics, secretome proteomics and plant pathogenicity assays. Nuclear Vta2 activates the expression of the adhesin-encoding yeast flocculin genes FLO1 and FLO11. Vta2 is required for fungal growth of Verticillium where it is a positive regulator of conidiation. Vta2 is mandatory for accurate timing and suppression of microsclerotia as resting structures. Vta2 controls expression of 270 transcripts, including 10 putative genes for adhesins and 57 for secreted proteins. Vta2 controls the level of 125 secreted proteins, including putative adhesins or effector molecules and a secreted catalase-peroxidase. Vta2 is a major regulator of fungal pathogenesis, and controls host-plant root infection and H2O2 detoxification. Verticillium impaired in Vta2 is unable to colonize plants and induce disease symptoms. Vta2 represents an interesting target for controlling the growth and development of these vascular pathogens.

Aspergillus oryzae is a safe mold widely used in food industry. It is also considered as a microbial cell factory for production of recombinant proteins and enzymes. Currently, genetic manipulation of filamentous fungi is achieved via Agrobacterium tumefaciens-mediated transformation methods usually employing antibiotic resistance markers. These methods are hardly usable for A. oryzae due to its strong resistance to the common antifungal compounds used for fungal transformation. In this study, we have constructed two binary vectors carrying the pyrG gene from A. oryzae as a biochemical marker than an antibiotic resistance marker, and an expression cassette for GFP or DsRed reporter gene under control of the constitutive gpdA promoter from Aspergillus nidulans. All components of these vectors are changeable to generate new versions for specific research purposes. The developed vectors are fully functional for heterologous expression of the GFP and DsRed fluorescent proteins in the uridine/uracil auxotrophic A. oryzae strain. Our study provides a new approach for A. oryzae transformation using pyrG as the selectable auxotrophic marker, A. tumefaciens as the DNA transfer tool and fungal spores as the transformation material. The binary vectors constructed can be used for gene expression studies in this industrially important filamentous fungus.

The plant-pathogenic fungus Verticillium longisporum is a causal agent of early senescence and ripening in cruciferous crops like Brassica napus. Verticillium wilts have become serious agricultural threats in recent decades. Verticillium species infect host plants through the roots and colonize xylem vessels of the host plant. The xylem fluid provides an environment with limited carbon sources and unbalanced amino acid supply, which requires V. longisporum to induce the cross-pathway control of amino acid biosynthesis. RNA-mediated gene silencing reduced the expression of the two CPC1 isogenes (VlCPC1-1 and VlCPC1-2) of the allodiploid V. longisporum up to 85%. VlCPC1 encodes the conserved transcription factor of the cross-pathway control. The silenced mutants were highly sensitive to amino-acid starvation, and the infected plants showed significantly fewer symptoms such as stunting or early senescence in oilseed rape plant infection assays. Consistently, deletion of single CPC1 of the haploid V. dahliae resulted in strains that are sensitive to amino-acid starvation and cause strongly reduced symptoms in the plant-host tomato (Solanum lycopersicum). The allodiploid V. longisporum and the haploid V. dahliae are the first phytopathogenic fungi that were shown to require CPC1 for infection and colonization of their respective host plants, oilseed rape and tomato.

The cruciferous fungal pathogen Verticillium longisporum represents an allodiploid hybrid with long spores and almost double the amount of nuclear DNA compared to other Verticillium species. V. longisporum evolved at least three times by hybridization. In Europe, virulent A1xD1 and avirulent A1xD3 hybrids were isolated from the oilseed crop Brassica napus. Parental A1 or D1 species are yet unknown whereas the D3 lineage represents Verticillium dahliae. Eleven V. longisporum isolates from Europe or California corresponding to hybrids A1xD1 or A1xD3 were compared. A single characteristic type of nuclear ribosomal DNA could be assigned to each hybrid lineage. The two avirulent A1xD3 isolates carried exclusively D3 ribosomal DNA (rDNA) which corresponds to V. dahliae. The rDNA of all nine A1xD1 isolates is identical but distinct from D3 and presumably originates from A1. Both hybrid lineages carry two distinct isogene pairs of four conserved regulatory genes corresponding to either A1 or D1/D3. D1 and D3 paralogues differ in several single nucleotide polymorphisms. Southern hybridization patterns confirmed differences between the A1 and D1/D3 isogenes and resulted in similar patterns for D1 and D3. Distinct signatures of the Verticillium transcription activator (VTA)2 regulatory isogene pair allow identification of V. longisporum hybrids by a single polymerase chain reaction and the separation from haploid species as V. dahliae or Verticillium albo-atrum. The combination between VTA2 signature and rDNA type identification represents an attractive diagnostic tool to discriminate allodiploid from haploid Verticillia and to distinguish between A1xD1 and A1xD3 hybrids which differ in their virulence towards B. napus. Electronic supplementary material The online version of this article (doi:10.1007/s00253-012-4530-1) contains supplementary material, which is available to authorized users.

The first leaky auxotrophic mutant for aromatic amino acids of the near-diploid fungal plant pathogen Verticillium longisporum (VL) has been generated. VL enters its host Brassica napus through the roots and colonizes the xylem vessels. The xylem contains little nutrients including low concentrations of amino acids. We isolated the gene Vlaro2 encoding chorismate synthase by complementation of the corresponding yeast mutant strain. Chorismate synthase produces the first branch point intermediate of aromatic amino acid biosynthesis. A novel RNA-mediated gene silencing method reduced gene expression of both isogenes by 80% and resulted in a bradytrophic mutant, which is a leaky auxotroph due to impaired expression of chorismate synthase. In contrast to the wild type, silencing resulted in increased expression of the cross-pathway regulatory gene VlcpcA (similar to cpcA/GCN4) during saprotrophic life. The mutant fungus is still able to infect the host plant B. napus and the model Arabidopsis thaliana with reduced efficiency. VlcpcA expression is increased in planta in the mutant and the wild-type fungus. We assume that xylem colonization requires induction of the cross-pathway control, presumably because the fungus has to overcome imbalanced amino acid supply in the xylem. Electronic supplementary material The online version of this article (doi:10.1007/s00253-009-2269-0) contains supplementary material, which is available to authorized users.