Spore Production Research Articles

Genotype-phenotype correlations within the Geodermatophilaceae.

The integration of genomic information into microbial systematics along with physiological and chemotaxonomic parameters provides for a reliable classification of prokaryotes. In silico analysis of chemotaxonomic traits is now being introduced to replace characteristics traditionally determined in the laboratory with the dual goal of both increasing the speed of the description of taxa and the accuracy and consistency of taxonomic reports. Genomics has already successfully been applied in the taxonomic rearrangement of Geodermatophilaceae (Actinomycetota) but in the light of new genomic data the taxonomy of the family needs to be revisited. In conjunction with the taxonomic characterisation of four strains phylogenetically located within the family, we conducted a phylogenetic analysis of the whole proteomes of the sequenced type strains and established genotype-phenotype correlations for traits related to chemotaxonomy, cell morphology and metabolism. Results indicated that the four isolates under study represent four novel species within the genus Blastococcus. Additionally, the genera Blastococcus, Geodermatophilus and Modestobacter were shown to be paraphyletic. Consequently, the new genera Trujillonella, Pleomorpha and Goekera were proposed within the Geodermatophilaceae and Blastococcus endophyticus was reclassified as Trujillonella endophytica comb. nov., Geodermatophilus daqingensis as Pleomorpha daqingensis comb. nov. and Modestobacter deserti as Goekera deserti comb. nov. Accordingly, we also proposed emended descriptions of Blastococcus aggregatus, Blastococcus jejuensis, Blastococcus saxobsidens and Blastococcus xanthilyniticus. In silico chemotaxonomic results were overall consistent with wet-lab results. Even though in silico discriminatory levels varied depending on the respective chemotaxonomic trait, this approach is promising for effectively replacing and/or complementing chemotaxonomic analyses at taxonomic ranks above the species level. Finally, interesting but previously overlooked insights regarding morphology and ecology were revealed by the presence of a repertoire of genes related to flagellum synthesis, chemotaxis, spore production and pilus assembly in all representatives of the family. A rich carbon metabolism including four different CO2 fixation pathways and a battery of enzymes able to degrade complex carbohydrates were also identified in Blastococcus genomes.

Comparison of one-step with two-step production of Bacillus atrophaeus spores for use as bioindicators.

The production method of spores significantly influences the resistance of spores used as bioindicators (BI) in the validation of sterilization of packaging material surfaces in aseptic food manufacturing. Therefore, the standardization of the spore production method represents an important and desirable goal in industrial BI production to ensure reliable validation test results. Previously, we recommended a two-step production approach for submerged spore production, in which the cultivation phase to obtain high cell mass was separate from the sporulation phase. In this work, a one-step manufacturing process was investigated to reduce production complexity and facilitate standardization of spore production. It was found that one-step BI production is technically possible but at the expense of spore yield. The two-step manufacturing process can realize almost 10-fold higher spore yields.

Characterization of key upstream asexual developmental regulators in Monascus ruber M7

Monascus species are traditional edible and medicinal filamentous fungi with a very long history in East Asia, but their sparse production of asexual spores (conidia) limits their large-scale industrial application. Previous studies have revealed that the flbA ortholog plays a negative role in the conidiation of Monascus ruber M7. FlbA, together with FlbB, FlbC, FlbD, FlbE, and FluG, were shown to be upstream development activators (UDAs) in the asexual development pathway in the model fungus Aspergillus nidulans. In the present study, homologs of these UDAs in M. ruber M7 were identified and functionally characterized. Disruption of MrflbB, MrflbD, and MrflbE delayed and decreased conidiation in M7, whereas the deletion of MrflbC or MrfluG resulted in no detectable change in sporulation. Deletion of MrflbA not only increased conidial number but also postponed sexual development in M7. Furthermore, the deletion of some UDAs also affected the production of secondary metabolites, particularly citrinin, whose yields were dramatically decreased in the ΔMrflbC and ΔMrflbA mutants. These findings provide new insights and opportunities to further explore the asexual development mechanism and modify the conidial production in Monascus spp.

Structural regulation and dynamic behaviour of organelles during plant meiosis.

Eukaryotes use various mechanisms to maintain cell division stability during sporogenesis, and in particular during meiosis to achieve production of haploid spores. In addition to establishing even chromosome segregation in meiosis I and II, it is crucial for meiotic cells to guarantee balanced partitioning of organelles to the daughter cells, to properly inherit cellular functions. In plants, cytological studies in model systems have yielded insights into the meiotic behaviour of different organelles, i.e., clearly revealing a distinct organization at different stages throughout meiosis indicating for an active regulatory mechanism determining their subcellular dynamics. However, how, and why plant meiocytes organize synchronicity of these elements and whether this is conserved across all plant genera is still not fully elucidated. It is generally accepted that the highly programmed intracellular behaviour of organelles during meiosis serves to guarantee balanced cytoplasmic inheritance. However, recent studies also indicate that it contributes to the regulation of key meiotic processes, like the organization of cell polarity and spindle orientation, thus exhibiting different functionalities than those characterized in mitotic cell division. In this review paper, we will outline the current knowledge on organelle dynamics in plant meiosis and discuss the putative strategies that the plant cell uses to mediate this programmed spatio-temporal organization in order to safeguard balanced separation of organelles. Particular attention is thereby given to putative molecular mechanisms that underlie this dynamic organelle organization taken into account existing variations in the meiotic cell division program across different plant types. Furthermore, we will elaborate on the structural role of organelles in plant meiosis and discuss on organelle-based cellular mechanisms that contribute to the organization and molecular coordination of key meiotic processes, including spindle positioning, chromosome segregation and cell division. Overall, this review summarizes all relevant insights on the dynamic behaviour and inheritance of organelles during plant meiosis, and discusses on their functional role in the structural and molecular regulation of meiotic cell division.

Characterization, Chemical Compounds and Biological Activities of Marrubium vulgare L. Essential Oil

As consumer trends shift towards more natural and ecological consumption patterns, industrialists are actively working towards substituting synthetic chemicals with natural and vegan products that contain bioactive properties. Thus, considering the shifts in customer demand and the growing concern around vegetable sourced productions, this work aims to contribute to the valorization of aromatic and medicinal Moroccan plants. By focusing on the Marrubium vulgare L. species, our objective is to carry out a physicochemical characterization to determine its chemical composition and biological activities. The volatile fraction collected by hydrodistillation (0.61%) and analyzed by GC-MS (gas chromatography coupled to mass spectrometry) contains five main compounds: 3-Thujanone, Eugenol, Topanol, Menthone and Piperitone. The antioxidant activity has been estimated by applying the DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging test and the ferric reducing antioxidant power (FRAP). The values of inhibitory concentration prove that our oil is a good antioxidant, with values of IC50 = 1.136 mg/mL and IC50 = 2.998 mg/mL, respectively, for the DPPH and FRAP tests. The results of the antifungal activity indicate a significant inhibition of mycelial growth for both tested molds, as well as a total inhibition of spore production at a concentration of 0.25 µL/mL.

Laboratory Evaluation of Temperature Effects on Germination, Radial Growth and Sporulation of Entomopathogenic Fungi and on Their Pathogenicity to Red Spider Mite, Tetranychus urticae Koch

Background: Entomopathogenic fungus (EPF) can provide safe and efficient insect pest management. Temperature, relative humidity (RH), light, air, nutritional content and host physiological condition are all elements that impact the proliferation and virulence of entomopathogenic fungus. The current study aimed to evaluate the effect of different temperatures on germination, growth, sporulation of entomopathogenic fungi and on their pathogenicity against Tetranychus urticae Koch was studied. Methods: Laboratory experiment was conducted to study the impact of temperature on germination, radial growth and sporulation of five entomopathogenic fungal isolates viz., B. bassiana (Bb 111), B. bassiana (Bb 112), B. bassiana (Bb 113), B. bassiana (Bb 114) and M. flavoviride var. minus. Virulence of five entomopathogenic fungal isolates subjected to different temperature against T. urticae also evaluated through laboratory bioassay techniques. Result: Results one valuation of entomopathogenic fungal isolates with four different temperatures at 20°C, 25°C, 30°C and 35°C showed that 25°C was optimum for germination, radial growth and sporulation. Maximum germination was recorded with the entomopathogenic isolate Beauveria bassiana (Bb 112) (95.67%) at 25°C. Interestingly, at 25°C all the B. bassiana isolates showed a radial growth of more than 50 mm with the highest recorded as 84.67 mm by B. bassiana (Bb 111). Maximum spore production was observed with B. bassiana (Bb 112) at 25°C (1.63 x 106 spores ml-1) followed by B. bassiana (Bb 111) (1.45 x 106 spores ml-1). It was also the most pathogenic against T. urticae at 25 and 30°C. The isolate Bb112 grown at 25°C caused higher mortality of 97.57 per cent against T. urticae. From the findings the isolates of B. bassiana (Bb 112) and (Bb 111) were found promising bio control agent against T. urticae with varying temperature.

Populations of Puccinia striiformis f. sp. tritici in Winter Spore Production Regions Spread from Southwestern Oversummering Areas in China.

Stripe rust, caused by Puccinia striifomis f. sp. tritici (Pst), is one of the most destructive wheat diseases in China. Understanding the interregional dispersal of Pst inoculum is important for controlling the disease. In the present study, wheat stripe rust samples collected from the winter spore production and oversummering regions in November 2018 to March 2019 were studied through virulence testing and molecular characterization. From 296 isolates, 96 races were identified using a set of 19 Chinese wheat cultivars and 111 races were identified using 18 Yr single-gene lines as differentials. The isolates from Hubei province in the winter spore production area had the highest similarity in virulence with those from eastern Yunnan in the oversummering area. Molecular characterization using 13 simple-sequence repeat and 43 Kompetitive allele specific PCR-single nucleotide polymorphism markers supported the conclusion that the Pst populations in the winter spore production regions were from Guizhou and eastern Yunnan, key oversummering areas in the southwest. Furthermore, an analysis of wind movement at the 700-hPa high altitude also supported the conclusion of spore dispersal from the southwestern oversummering region to the south-central winter spore production region. The results of this study provide an epidemiological basis for deploying various effective resistance genes in different regions to control stripe rust.

Dry-Season Soil and Co-Cultivated Host Plants Enhanced Propagation of Arbuscular Mycorrhizal Fungal Spores from Sand Dune Vegetation in Trap Culture.

The use of arbuscular mycorrhizal fungi (AMF) as biofertilizer in agriculture is a sustainable approach to fertilization. The first step in the production of AMF biofertilizer is inoculation of mycotrophic plants with a composite of soil and native plant roots, containing potentially viable AMF spores from natural habitats, to a trap culture. A single host plant or a consortium of host plants can be used to propagate AMF spores. However, the difference in the comparative efficiency of mono- and co-cultivated host plants used for the production of AMF spores and the maintenance of original AMF community composition has not been well elucidated. Here, we prepared trap culture with nutrient-poor soil from coastal sand dune vegetation collected during the dry season when the AMF spore density and relative abundance of Glomeromycota ITS2 sequences were significantly higher (p = <0.05) than in the wet season. The AMF communities in the soil were mainly composed of Glomus spp. Maize (Zea mays L.) and/or Sorghum (Sorghum bicolor (L.). Moench) were grown in trap cultures in the greenhouse. Our results demonstrated that co-cultivation of the host plants increased the production of AMF spores but, compared to mono-cultivation of host plants, did not better sustain the native AMF community compositions in the coastal sand dune soil. We propose that the co-cultivation of host plants in a trap culture broadens AMF-host plant compatibilities and thus sustains the symbiotic association of the natively diverse AMF. Therefore, the results of this study suggest that further research is needed to confirm whether the co-culturing of more than one host plant is as efficient a strategy as using a monoculture of a single host plant.

The role of Acpbs2 in the asexual sporulation, stress responseand carbon metabolism of Aspergillus cristatus.

Aspergillus cristatus is the dominant fungus during the fermentation of Fuzhuan brick tea, hypotonic conditions only induced its sexual development to produce ascospores, while hypertonic conditions only induced its asexual development to produce conidia, indicating that osmotic stress can regulate spore production in A. cristatus. However, the underlying regulatory mechanism is unclear. In this study, the roles of Acpbs2, which is homologous to pbs2 from Saccharomyces cerevisiae, in sporulation, stress responses, the color of colonies, and carbon metabolism were explored in A. cristatus. Deletion mutants of Acpbs2 were obtained by homologous recombination. The time required to produce conidia was delayed, and the number of conidia produced was significantly reduced in hypertonic media in ΔAcpbs2 by phenotypic observations, indicating that Acpbs2 plays a positive role in asexual development. Stress sensitivity tests showed that the order of the sensitivity of ΔAcpbs2 to different osmotic regulators was 3 M NaCl > 3 M sucrose > 3 M sorbitol. Moreover, the deletion mutants were sensitive to high oxidative stress. The growth of the Acpbs2 deletion mutant was inhibited under alkaline-pH stress, indicating that Acpbs2 is involved in high pH stress tolerance. Additionally, compared with the wild type, the colony color of the Acpbs2 deletion mutant became lighter. All the above developmental defects were reversed by the reintroduction of the Acpbs2 gene in ΔAcpbs2. Transcriptome data showed that Acpbs2 regulated the expression of several genes related to conidial development, osmotic stress, oxidative stress, and carbon metabolism. More importantly, the interaction between Acpbs2 and its downstream gene Achog1 was verified by yeast two-hybrid assays. We speculated that this interaction might regulate the osmotic stress response, the oxidative stress response, and asexual sporulation in A. cristatus, which will be one of the focuses of our future research.

Identification of three Diaporthe species from Selenicereus sp. explants and their pathogenicity to three dragon fruit species

AbstractDragon fruit (Selenicereus spp.) is a cash fruit crop worldwide. One of the most effective techniques in producing clean planting materials and preventing disease infection is a plant tissue culture system. This study investigated the identity and characteristics of fungal contaminants in tissue‐cultured red‐fleshed dragon fruit (S. monacanthus) stem explants. Four fungal isolates were recovered from contaminated S. monacanthus cultures. The fungal isolates showed thick and white to greyish mycelia 7 days after incubation (dai) in potato dextrose agar medium with spore production 13 dai. The isolates were identified as Diaporthe sp., based on combined morphological and cultural characteristics. DNA analysis of the partial sequence of the ribosomal internal transcribed spacer (ITS), beta‐tubulin 2 (TUB) and transcription elongation factor 1‐α (TEF1‐α) gene regions revealed a high similarity of the isolates to D. passifloricola, D. limonicola and D. tectonae. In detached stem and glasshouse assays, the three Diaporthe species identified were found pathogenic to three dragon fruit species (S. megalanthus, S. undatus and S. monacanthus). Early symptoms observed were similar to the disease of dragon fruit (caused by the same fungal genus) previously reported in Malaysia. The fungi were re‐isolated from the inoculated stems, establishing Koch's postulate. This study confirms three Diaporthe species as contaminants in dragon fruit explants, reports the susceptibility of S. monacanthus and S. undatus to a stem disease, and the tolerance of S. megalanthus to the disease caused by Diaporthe species.

Colony growth and biofilm formation of Aspergillus niger under simulated microgravity.

The biotechnology- and medicine-relevant fungus Aspergillus niger is a common colonizer of indoor habitats such as the International Space Station (ISS). Being able to colonize and biodegrade a wide range of surfaces, A. niger can ultimately impact human health and habitat safety. Surface contamination relies on two key-features of the fungal colony: the fungal spores, and the vegetative mycelium, also known as biofilm. Aboard the ISS, microorganisms and astronauts are shielded from extreme temperatures and radiation, but are inevitably affected by spaceflight microgravity. Knowing how microgravity affects A. niger colony growth, in particular regarding the vegetative mycelium (biofilm) and spore production, will help prevent and control fungal contaminations in indoor habitats on Earth and in space. Because fungal colonies grown on agar can be considered analogs for surface contamination, we investigated A. niger colony growth on agar in normal gravity (Ground) and simulated microgravity (SMG) conditions by fast-clinorotation. Three strains were included: a wild-type strain, a pigmentation mutant (ΔfwnA), and a hyperbranching mutant (ΔracA). Our study presents never before seen scanning electron microscopy (SEM) images of A. niger colonies that reveal a complex ultrastructure and biofilm architecture, and provide insights into fungal colony development, both on ground and in simulated microgravity. Results show that simulated microgravity affects colony growth in a strain-dependent manner, leading to thicker biofilms (vegetative mycelium) and increased spore production. We suggest that the Rho GTPase RacA might play a role in A. niger’s adaptation to simulated microgravity, as deletion of ΔracA leads to changes in biofilm thickness, spore production and total biomass. We also propose that FwnA-mediated melanin production plays a role in A. niger’s microgravity response, as ΔfwnA mutant colonies grown under SMG conditions showed increased colony area and spore production. Taken together, our study shows that simulated microgravity does not inhibit A. niger growth, but rather indicates a potential increase in surface-colonization. Further studies addressing fungal growth and surface contaminations in spaceflight should be conducted, not only to reduce the risk of negatively impacting human health and spacecraft material safety, but also to positively utilize fungal-based biotechnology to acquire needed resources in situ.

A Botybirnavirus Isolated from Alternaria tenuissima Confers Hypervirulence and Decreased Sensitivity of Its Host Fungus to Difenoconazole

Alternaria alternata botybirnavirus 1 (AaBRV1) was isolated from a strain of Alternaria alternata, causing watermelon leaf blight in our previous research. The effect of AaBRV1 on the phenotype of its host fungus, however, was not determined. In the present study, a novel strain of AaBRV1 was identified in A. tenuissima strain TJ-NH-51S-4, the causal agent of cotton Alternaria leaf spot, and designated as AaBRV1-AT1. A mycovirus AaBRV1-AT1-free strain TJ-NH-51S-4-VF was obtained by protoplast regeneration, which eliminated AaBRV1-AT1 from the mycovirus AaBRV1-AT1-infected strain TJ-NH-51S-4. Colony growth rate, spore production, and virulence of strain TJ-NH-51S-4 were greater than they were in TJ-NH-51S-4-VF, while the sensitivity of strain TJ-NH-51S-4 to difenoconazole, as measured by the EC50, was lower. AaBRV1-AT1 was capable of vertical transmission via asexual spores and horizontal transmission from strain TJ-NH-51S-4 to strain XJ-BZ-5-1hyg (another strain of A. tenuissima) through hyphal contact in pairing cultures. A total of 613 differentially expressed genes (DEGs) were identified in a comparative transcriptome analysis between TJ-NH-51S-4 and TJ-NH-51S-4-VF. Relative to strain TJ-NH-51S-4-VF, the number of up-regulated and down-regulated DEGs in strain TJ-NH-51S-4 was 286 and 327, respectively. Notably, the expression level of one DEG-encoding cytochrome P450 sterol 14α-demethylase and four DEGs encoding siderophore iron transporters were significantly up-regulated. To our knowledge, this is the first documentation of hypervirulence and reduced sensitivity to difenoconazole induced by AaBRV1-AT1 infection in A. tenuissima.

A New Strain of Lecanicillium uredinophilum Isolated from Tibetan Plateau and Its Insecticidal Activity.

A new strain QHLA of Lecanicillium uredinophilum was isolated from a Chinese caterpillar fungus complex and its optimum growth temperature and fermentation conditions were studied. Its insecticidal activity was tested against larvae of seven different insect pests, including Henosepilachna vigintioctopunctata, Spodoptera exigua, Plutella xylostella, Spodoptera frugiperda, Sitobion avenae, Hyalopterus perikonus, and Aphis citricola. The optimum growth temperature was 21–24 °C. The highest spore production of the strain QHLA was 4.08 × 106 spore/mL on solid medium with a nitrogen source of NH4Cl. However, the highest mycelial growth rate of the strain QHLA was on solid medium with a nitrogen source from yeast extract (4.63 ± 0.03 mm/d). When the liquid medium contained peptone, yeast extract, and glucose, the water content of the mycelia was the lowest, while the spore production was the highest until day 12. When the liquid medium contained glucose, tussah pupa powder, KH2PO4, and MgSO4, the mycelia production was highest until day 8. The bioassay for insecticidal activity indicated that the LC50 values of QHLA were 6.32 × 103 spore/mL and 6.35 × 103 spore/mL against Sitobion avenae and Aphis citricola, respectively, while the LC90 values of the strain QHLA against Aphis citricola and Sitobion avenae were 2.11 × 107 spore/mL and 2.36 × 108 spore/mL, respectively. Our results demonstrated that the strain QHLA was a high virulence pathogenic fungus against insect pests, with the potential to be developed as a microbial pesticide.

Trichoderma harzianum sensu lato TSM39: A wheat microbiome fungus that mitigates spot blotch disease of wheat (Triticum turgidum L. subsp. durum) caused by Bipolaris sorokiniana

Bipolaris sorokiniana - the causal agent of wheat spot blotch - is an emerging phytopathogenic fungus in the Yaqui Valley, Sonora, Mexico. At present, its control relies on the use of harmful chemicals, partly due to the lack of effective alternatives. Thus, this work aimed to identify and characterize indigenous wheat-associated fungal strains with biocontrol capacity against B. sorokiniana that can be integrated into control strategies for spot blotch management. Out of 59 fungal strains evaluated, we identified 5 strains with antagonistic activity against B. sorokiniana by dual confrontation assay, and these were taxonomically assigned as Trichoderma harzianum sensu lato (strains TSM39 and TSO39), Aspergillus terreus (strains TRQ89 and TRQ93), and Aspergillus nidulans (strain TRQ83) based on phylogenetic analysis using the ITS region of the rRNA cistron. Trichoderma harzianum TSM39 showed interesting biological control traits (i.e., strong antagonistic activity, high growth rate, non-cytotoxicity) and its application to wheat plants - under highly conducive conditions for spot blotch outbreak- resulted in a significant reduction of disease severity. In vitro experiments revealed that the chitinolytic system of strain TSM39 responds to B. sorokiniana lysates (signals), which could have potential biotechnological applications to improve its biocontrol activity. Additionally, a solid-state fermentation system was successfully established for spore production of strain TSM39. Taken together, Trichoderma harzianum sensu lato TSM39 is a biocontrol agent against B. sorokiniana with the potential to be integrated into spot blotch management strategies and contribute to the sustainability of the region.

Selecting alternative sterile and non-sterile substrates for mycorrhizal inoculant production

Alternative substrates were evaluated for mycorrhizal inoculants production considering the beneficial effects of arbuscular mycorrhizal fungi (AMF) for plants and environment. The first step of the experiment aimed at evaluating the potential of substrates produced from sugarcane bagasse (SB), coconut fibre (CF) and urban waste compost (UWC) to produce AMF inoculants, by using Brachiaria decumbens as multiplier plant. The experiment has followed a randomized block design, at 7x3 factorial arrangement (seven substrates: SB, CF, SB+CF 1:1, SB+UWC 3:1, CF+UWC 3:1, SB+CF+UWC 3:3:2, Soil+Sand 3:1, vs. three substrate preparations: inoculated/sterilized, inoculated/non-sterilized, non-inoculated/sterilized). Inoculums produced in the first experimental stage were tested at the second stage, based on the inoculated/sterilized preparation. Seed germination, dry shoot biomass, mycorrhizal root rate and total spore production were assessed in both tests. There was significant decrease in seed emergence in organic substrates in relation to Soil+Sand, although the plants growth in organic substrates was greater. Artificial inoculation was not superior to spontaneous substrate mycorrhization or affected inoculum production. Mycorrhizal colonization of roots was equivalent in most organic substrates and in Soil+Sand. However, spore production stood out in organic substrate mixes, mainly when CF and UWC were used. This finding was confirmed at the second experimental stage.

Characterization of the virulence of three novel clade 2 Clostridioides (Clostridium) difficile strains and a two-year screening in animals and humans in Brazil.

Clostridioides (Clostridium) difficile infection (CDI) is an evolving global healthcare problem, and owing to the diverse and dynamic molecular epidemiology of C. difficile, new strains continue to emerge. In Brazil, only two cases of CDI due to the so called hypervirulent PCR ribotype (RT) 027 belonging to clade 2 have ever been reported, whereas incidence of CDI due to another “hypervirulent” RT078 (clade 5) has not yet been reported. In contrast, novel clade 2 strains have been identified in different hospitals. To better understand the epidemiology of CDIs in Brazil, this study aimed to genotypically and phenotypically characterize three novel Brazilian clade 2 strains (RT883, 884, and 885) isolated from patients with confirmed CDI. In addition, to better understand the circulating RTs, a two-year sampling was conducted in patients from the same hospital and in several domestic and wild animal species. The three strains examined showed lower production of A/B toxins than the control RT027, although two of these strains harbored a truncated tcdC gene. All strains showed swimming motility similar to that of RT027, while RT883 showed higher spore production than the reference strain. In the in vivo hamster model, the lethality of all strains was found to be similar to that of RT027. Both cgMLST and cgMLSA analyses revealed a high genetic similarity among the three-novel clade 2 isolates. In the two-year survey in animals and humans, RT883, 884, and 885 were not detected; however, three new RTs (RT988, RT989, and RT990) were isolated, two of which were genetically related to the three previously reported clade 2 strains. RT106 and RT126 were most frequently detected in humans (47.9%) and animals (57.9%), respectively. Furthermore, RT027 and RT078 were not detected in humans. The results of this study suggest that these novel clade 2 strains have virulence potential and that new strains from clade 2 continue to emerge in our setting, indicating the need for long-term local surveillance.

Evolving social behavior through selection of single-cell adhesion in Dictyostelium discoideum

SummaryThe social amoeba Dictyostelium discoideum commonly forms chimeric fruiting bodies. Genetic variants that produce a higher proportion of spores are predicted to undercut multicellular organization unless cooperators assort positively. Cell adhesion is considered a primary factor driving such assortment, but evolution of adhesion has not been experimentally connected to changes in social performance. We modified by experimental evolution the efficiency of individual cells in attaching to a surface. Surprisingly, evolution appears to have produced social cooperators irrespective of whether stronger or weaker adhesion was selected. Quantification of reproductive success, cell-cell adhesion, and developmental patterns, however, revealed two distinct social behaviors, as captured when the classical metric for social success is generalized by considering clonal spore production. Our work shows that cell mechanical interactions can constrain the evolution of development and sociality in chimeras and that elucidation of proximate mechanisms is necessary to understand the ultimate emergence of multicellular organization.

Efecto de los niveles de salinidad sobre la actividad antifúngica del aceite esencial de Thymus contra Fusarium oxysporum

Thyme (Thymus sp.), a medicinal plant of the family Lamiaceae, is used in traditional medicine, contains a wide array of medicinally active components, in their great majority of a rather complex mixture of thymol, ρ-Cymene, γ-Terpinene, β-Caryophyllen, etc. This study aimed to evaluate the efficacy of Thymus vulgaris extract against Fusarium oxysporum f. sp.radicis-lycopersici strain under saline conditions, assuming soil with high salt content of the arid regions. Essential oil was extracted by hydrodistillation technique using a Clevenger apparatus. The essential oil compounds were identified by GC-MS analysis. Antifungal activity of essential oil against Fusarium oxysporum f. sp. radicis-lycopersici was investigated by agar dilution method. The main constituents of thyme essential oil were thymol (76.96 %), ρ-cymene (9.89 %) and γ-Terpinene (1.92 %). Essential oil from Thymus resented high in vitro activity, in controlling conidial germination and mycelial growth. However, the oil was significantly not active against the spore production under a salinity medium. The results showed that mycelial growth was stimulated in concentrations with 0.6 -1.5 %. In contrast, it was significantly reduced at a higher concentration (2 %). The application of NaCl caused a significant increase in the conidia production at various concentrations tested. NaCl has a minor inhibitory effect on conidial germination only when the concentration was 2 %. The results of this study indicate that salinity decreases the efficacy of essential oil against the pathogen.

In vitro antifungal activity of alcohol and aqueous extract of stachys sp on fusarium solani

The genus Stachys L. (Lamiaceae) includes about 300 species as annual or perennial herbs or small shrubs, spread in temperate regions of Mediterranean, Asia, America and southern Africa. Stachys sp has antioxidant activity and destroys free radicals. Antifungal effects of methanolic extracts of four different types of Stachys on several bacteria and yeasts were recorded. This study was conducted to evaluate the effect of the alcoholic and aqueous extract of Stachys sp. on the growth and spore viability of Fusarium solani and the results revealed that the extract affect the daily growth of Fusarium solani and the effect was increased with the increasing the concentration. The lowest rate of daily growth was recorded in the case alcohol extract which was 0.203cm at the concentration 25% while the highest one was 0.537cm at the concentration 6.25% as compared with the control 1.00cm. The aqueous and alcohol extract of Stachys sp suppressed the spore production (sporogenesis) of Fusarium significantly as compared with the control and the effect increased with the increasing of concentration.

Systematic stepwise screening of new microbial antagonists for biological control of European canker

Neonectria ditissima is the causal agent of European canker. This pathogen causes cankers on apple branches and the main stem, which may lead to the loss of the whole tree. Chemical control is the essential component in disease management and no suitable biocontrol products are commercially available. This study aimed at selecting potential microbial antagonists against N. ditissima through a systematic stepwise screening program for the development of a new biocontrol product. A total of 520 potential candidates were isolated from apple branches showing canker symptoms. Important characteristics for application of Microbial Biological Control Agents were tested per each candidate: spore production, spore survival during storage, cold tolerance, drought tolerance and UV-B resistance. Isolates able to germinate and grow at human body temperature were excluded. A total of 252 candidates fulfilled the stablished criteria. All 520 candidates belonged to 44 different taxonomic groups, being the most abundant Alternaria spp. (22.2 %), Aureobasidium pullulans (16.1 %), Cladosporium spp. (9.5 %) and Fusarium spp. (9.0 %). Information on possible pathogenicity and toxicity for humans, animals, plants and the environment and on patents in biocontrol use was collected per each identified species. A total of 158 candidates belonging to species that did not show potential risks or patent conflicts were assessed by their antagonistic behaviour against N. ditissima in bioassays in planta. Each candidate was inoculated in ‘Elstar’ apple branches inoculated with N. ditissima 24 h before. Inoculated branches were incubated at 17 °C, 16 h light per day and RH > 90 %. After four weeks, canker symptom expression was visually assessed. The capacity of the candidates to reduce colonisation of N. ditissima in the branches was evaluated by quantifying N. ditissima DNA concentration using qPCR. Four candidates of Clonostachys rosea showed antagonistic properties; after four weeks of treatment, no canker symptoms or small bark cracks were observed in the inoculated branches and N. ditissima DNA was reduced by 90–99 %. Following them, the branches inoculated with one candidate of Akanthomyces muscarius, A. pullulans and Cladosporium europaeum showed small bark cracks and small swollen bark and N. ditissima DNA was reduced by more than 90 %. The systematic stepwise screening approach was a powerful strategy to find new MBCAs against N. ditissima with antagonistic properties that also fulfilled major criteria with regards to commercial production and safety, as well as ecological needs.