Foliose Ulva Species Show Considerable Inter‐Specific Genetic Diversity,Low Intra‐Specific Genetic Variation,and the Rare Occurrence of Inter‐Specific Hybrids in the Wild

Foliose Ulva spp. have become increasingly important worldwide for their environmental and financial impacts. A large number of such Ulva species have rapid reproduction and proliferation habits, which explains why they are responsible for Ulva blooms, known as “green tides”, having dramatic negative effects on coastal ecosystems, but also making them attractive for aquaculture applications. Despite the increasing interest in the genus Ulva, particularly on the larger foliose species for aquaculture, their inter‐ and intra‐specific genetic diversity is still poorly described. We compared the cytoplasmic genome (chloroplast and mitochondrion) of 110 strains of large distromatic foliose Ulva from Ireland, Brittany (France), the Netherlands and Portugal. We found six different species, with high levels of inter‐specific genetic diversity, despite highly similar or overlapping morphologies. Genetic variation was as high as 82 SNPs/kb between Ulva pseudorotundata and U. laetevirens, indicating considerable genetic diversity. On the other hand, intra‐specific genetic diversity was relatively low, with only 36 variant sites (0.03 SNPs/kb) in the mitochondrial genome of the 29 Ulva rigida individuals found in this study, despite different geographical origins. The use of next‐generation sequencing allowed for the detection of a single inter‐species hybrid between two genetically closely related species, U. laetevirens, and U. rigida, among the 110 strains analyzed in this study. Altogether, this study represents an important advance in our understanding of Ulva biology and provides genetic information for genomic selection of large foliose strains in aquaculture.     

Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.     

Species‐Specific Barriers to Tree Regeneration in High Elevation Habitats of West Virginia

Herbaceous competition and herbivory have been identified as critical barriers to restoration of native tree species in degraded landscapes around the world; however, the combined effects of competition and herbivory are poorly understood. We experimentally manipulated levels of herbivory and herbaceous competition and analyzed the response of tree seedling performance over three growing seasons as a function of species and habitat in north‐central West Virginia. Four native tree species were planted in old field and forest experimental plots: Castanea dentata (American chestnut), Quercus rubra (red oak), Acer saccharum (sugar maple), and Picea rubens (red spruce). Red spruce demonstrated the highest growth increment and greatest survival (64%) and most consistent results among treatments and habitats. Red spruce survival was not reduced in the presence of Odocoileus virginianus (white‐tailed deer) browse and herbaceous competition; however, growth was improved by suppression of herbaceous competition. We suspect that this deciduous forest landscape would regenerate to a red spruce dominated forest if seed source was available. In contrast, the other three species tested had very low survival when exposed to deer and were more responsive to competing vegetation and habitat type. American chestnut had low survival and growth across all treatments, suggesting basic climate limitations. Vigorous natural regeneration of Prunus serotina (black cherry) occurred in forest plots where both competing herbs and deer were excluded. Our results demonstrated the importance of testing multiple potential recruitment barriers and species at once and the need for species and habitat‐specific restoration treatments.     

Specific and Rapid Detection of Lily symptomless virus and Arabis mosaic virus in Lily by Dual IC‐RT‐PCR

Lily symptomless virus (LSV) and Arabis mosaic virus (ArMV) cause severe losses of quantity and quality of lily flower and bulb production. Specificity, sensitivity and speed of detection methods for viruses need to be improved greatly to prevent LSV and ArMV from spreading from infected lilies. A dual IC‐RT‐PCR procedure for detection was developed in which the antibodies of LSV and ArMV were mixed and the mixture used to coat the PCR tubes. The particles of the two viruses were captured by the respective antibodies. Interference by other RNA viruses in infected lily was eliminated in the RT‐PCR. Also, an RNA extraction step was omitted. The dual IC‐RT‐PCR products of LSV and ArMV were 521 bp and 691 bp, respectively. The specificity of the method was validated; only LSV and ArMV of four viruses were detected by dual IC‐RT‐PCR. The sensitivity of the detection method is 1 mg leaf tissue and higher than DAS‐ELISA due to enrichment by dual immunocapture.     

Immunoassay‐based Techniques for Early and Specific Detection of Latent Postharvest Anthracnose in Mango

The postharvest anthracnose pathogen Colletotrichum gloeosporioides inciting latent or quiescent infection of mango was detected in early stages using immunoassay methods. Twenty‐five pathotypes isolated from different agroclimatic zones of Tamil Nadu, Karnataka and Pondicherry, India, revealed the variation in protein profile analysis (SDS‐PAGE). The polyclonal antibodies (PCA) were raised against the unfractioned mycelial protein (UMP) and a 40‐kDa polypeptide present in all pathotypes. Standardization of antigen and antiserum dilutions revealed that an antigen dilution of 1 : 200 (protein concentration of 20 μg/ml) and antiserum dilution of 1 : 100 (protein concentration of 40 μg/ml raised against UMP) and 1 : 200 (protein concentration of 20 μg/ml raised against 40 kDa polypeptide) was found to be optimum for the detection of anthracnose pathogen. Both antisera detected the C. gloeosporioides antigen in enzyme‐linked immunosorbent assays (ELISAs), dot immunobinding assays (DIBAs) and Western blots. The specificity in reaction was compared by isolating other Colletotrichum spp. from various hosts viz., C. lindemuthianum (beans), C. falcatum (sugarcane), C. musae (banana), C. capsici (chillies) and Botryodiplodia theobromae (mango). The antisera generated against UMP revealed the cross‐reaction with other host isolates and mango stem end rot pathogen (B. theobromae). The PCA raised against 40‐kDa polypeptide exhibited the specific reaction with C. gloeosporioides isolates in all the immunoassay techniques. By utilizing both PCA, the presence of latent infection was observed in healthy‐looking leaves, flowers and fruits in orchard conditions. The fruit tissues recorded high absorbance values followed by flowers and leaves in all the detection methods. The ELISA technique was also useful in assessing the pathogen inoculum at various biocontrol formulations sprayed mango trees under field conditions. The fluorescent pseudomonad strains mixture (KFP1 + FP7) amended with chitin sprayed at 30‐day intervals revealed the significant reduction in pathogen load than other formulations and unsprayed control.     

Cues of Predation Risk Induce Instar‐ and Genotype‐Specific Changes in Pea Aphid Colony Spatial Structure

Deploying collective antipredator behaviors during periods of increased predation risk is a major determinant of individual fitness for most animal groups. Pea aphids, Acyrthosiphon pisum, which live in aggregations of genetically identical individuals produced via asexual reproduction warn nearby conspecifics of pending attack by secreting a volatile alarm pheromone. This alarm pheromone allows clone‐mates to evade predation by walking away or dropping off the host plant. Here, we test how a single alarm pheromone emission influences colony structure and defensive behavior in this species. Relative to control colonies, groups exposed to alarm pheromone exhibited pronounced escape behavior where many individuals relocated to adjacent leaves on the host plant. Alarm pheromone reception, however, also had subtle instar‐specific effects: The proportion of 1st instars feeding nearest the leaf petiole decreased as these individuals moved to adjacent leaves, while the proportion of 2nd–3rd instars feeding nearest the leaf petiole remained constant. Fourth instars also dispersed to neighboring leaves after pheromone exposure. Lastly, alarm pheromone reception caused maternal aphids to alter their preferred feeding sites in a genotype‐specific manner: Maternal aphids of the green genotype fed further from the petiole, while maternal aphids of the pink genotype fed closer to the petiole. Together, our results suggest that aphid colony responses to alarm pheromone constitute a diversity of nuanced instar‐ and genotype‐specific effects. These behavioral responses can dramatically change the spatial organization of colonies and their collective defensive behavior.     

Specific protein homeostatic functions of small heat‐shock proteins increase lifespan

During aging, oxidized, misfolded, and aggregated proteins accumulate in cells, while the capacity to deal with protein damage declines severely. To cope with the toxicity of damaged proteins, cells rely on protein quality control networks, in particular proteins belonging to the family of heat‐shock proteins (HSPs). As safeguards of the cellular proteome, HSPs assist in protein folding and prevent accumulation of damaged, misfolded proteins. Here, we compared the capacity of all Drosophila melanogaster small HSP family members for their ability to assist in refolding stress‐denatured substrates and/or to prevent aggregation of disease‐associated misfolded proteins. We identified CG14207 as a novel and potent small HSP member that exclusively assisted in HSP70‐dependent refolding of stress‐denatured proteins. Furthermore, we report that HSP67BC, which has no role in protein refolding, was the most effective small HSP preventing toxic protein aggregation in an HSP70‐independent manner. Importantly, overexpression of both CG14207 and HSP67BC in Drosophila leads to a mild increase in lifespan, demonstrating that increased levels of functionally diverse small HSPs can promote longevity in vivo.     

State‐of‐the‐art CRISPR/Cas9 Technology for Genome Editing in Trypanosomatids

CRISPR/Cas9 technology has revolutionized biology. This prokaryotic defense system against foreign DNA has been repurposed for genome editing in a broad range of cell tissues and organisms. Trypanosomatids are flagellated protozoa belonging to the order Kinetoplastida. Some of its most representative members cause important human diseases affecting millions of people worldwide, such as Chagas disease, sleeping sickness and different forms of leishmaniases. Trypanosomatid infections represent an enormous burden for public health and there are no effective treatments for most of the diseases they cause. Since the emergence of the CRISPR/Cas9 technology, the genetic manipulation of these parasites has notably improved. As a consequence, genome editing is now playing a key role in the functional study of proteins, in the characterization of metabolic pathways, in the validation of alternative targets for antiparasitic interventions, and in the study of parasite biology and pathogenesis. In this work we review the different strategies that have been used to adapt the CRISPR/Cas9 system to Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp., as well as the research progress achieved using these approaches. Thereby, we will present the state‐of‐the‐art molecular tools available for genome editing in trypanosomatids to finally point out the future perspectives in the field.     

Antipredator Behavior in Desmognathus ochrophaeus: Threat‐Specific Responses to Chemical Stimuli in a Foraging Context

Prey species may reduce the likelihood of injury or death by engaging in defensive behavior but often incur costs related to decreased foraging success or efficiency. To lessen these costs, prey may adjust the intensity or type of antipredator behavior according to the nature of the perceived threat. We evaluated the potential for threat‐sensitive responses by Allegheny Mountain dusky salamanders (Desmognathus ochrophaeus) exposed to chemical stimuli associated with predation by asking three questions: (1) Do individual D. ochrophaeus respond to chemical cues in a threat‐sensitive manner? (2) Do salamanders exhibit the same pattern of behavioral response while foraging? and (3) Is foraging efficiency reduced when focal individuals are exposed to stimuli from predators or predation events? In our first experiment, we evaluated salamander chemosensory movements (nose‐taps), locomotor activity (steps), and edge behavior in response to chemical stimuli from disturbed and injured conspecifics as well as predatory Gyrinophilus porphyriticus and found that individual D. ochrophaeus show a significant graded increase in nose‐taps when exposed to cues from conspecifics and a reduction in activity when exposed to the predator. In our second experiment, we again observed salamander responses to the same chemical stimuli but in this instance added five Drosophila prey to the test dishes. We found that salamanders exhibited a similar pattern of response to the chemical stimuli in the presence of prey, showing a graded increase in nose‐taps to cues from conspecifics and a reduction in activity when exposed to the predator. However, foraging efficiency (i.e. the proportion of successful strikes) did not vary significantly among treatments. Our data show that individual D. ochrophaeus detect and differentially respond to chemical stimuli associated with predation, but do not significantly reduce foraging efficiency. Overall, the type and relative intensity of these responses is largely unaffected by the presence of potential prey.     

The diversion of 2‐C‐methyl‐d‐erythritol‐2,4‐cyclodiphosphate from the 2‐C‐methyl‐d‐erythritol 4‐phosphate pathway to hemiterpene glycosides mediates stress responses in Arabidopsis thaliana

2‐C‐Methyl‐d ‐erythritol‐2,4‐cyclodiphosphate (MEcDP) is an intermediate of the plastid‐localized 2‐C‐methyl‐d ‐erythritol‐4‐phosphate (MEP) pathway which supplies isoprenoid precursors for photosynthetic pigments, redox co‐factor side chains, plant volatiles, and phytohormones. The Arabidopsis hds‐3 mutant, defective in the 1‐hydroxy‐2‐methyl‐2‐(E)‐butenyl‐4‐diphosphate synthase step of the MEP pathway, accumulates its substrate MEcDP as well as the free tetraol 2‐C‐methyl‐d ‐erythritol (ME) and glucosylated ME metabolites, a metabolic diversion also occurring in wild type plants. MEcDP dephosphorylation to the free tetraol precedes glucosylation, a process which likely takes place in the cytosol. Other MEP pathway intermediates were not affected in hds‐3. Isotopic labeling, dark treatment, and inhibitor studies indicate that a second pool of MEcDP metabolically isolated from the main pathway is the source of a signal which activates salicylic acid induced defense responses before its conversion to hemiterpene glycosides. The hds‐3 mutant also showed enhanced resistance to the phloem‐feeding aphid Brevicoryne brassicae due to its constitutively activated defense response. However, this MEcDP‐mediated defense response is developmentally dependent and is repressed in emerging seedlings. MEcDP and ME exogenously applied to adult leaves mimics many of the gene induction effects seen in the hds‐3 mutant. In conclusion, we have identified a metabolic shunt from the central MEP pathway that diverts MEcDP to hemiterpene glycosides via ME, a process linked to balancing plant responses to biotic stress.     

Amplicon‐pyrosequencing‐based detection of compositional shifts in bryophyte‐associated fungal communities along an elevation gradient

Although bryophytes are a dominant vegetation component of boreal and alpine ecosystems, little is known about their associated fungal communities. HPLC assays of ergosterol (fungal biomass) and amplicon pyrosequencing of the ITS2 region of rDNA were used to investigate how the fungal communities associated with four bryophyte species changed across an elevational gradient transitioning from conifer forest to the low‐alpine. Fungal biomass and OTU richness associated with the four moss hosts did not vary significantly across the gradient (P > 0.05), and both were more strongly affected by host and tissue type. Despite largely constant levels of fungal biomass, distinct shifts in community composition of fungi associated with Hylocomium, Pleurozium and Polytrichum occurred between the elevation zones of the gradient. This likely is a result of influence on fungal communities by major environmental factors such as temperature, directly or indirectly mediated by, or interacting with, the response of other components of the vegetation (i.e. the dominant trees). Fungal communities associated with Dicranum were an exception, exhibiting spatial autocorrelation between plots, and no significant structuring by elevation. Nevertheless, the detection of distinct fungal assemblages associated with a single host growing in different elevation zones along an elevational gradient is of particular relevance in the light of the ongoing changes in vegetation patterns in boreal and alpine systems due to global climate warming.     

HT proteins contribute to S‐RNase‐independent pollen rejection in Solanum

Plants have mechanisms to recognize and reject pollen from other species. Although widespread, these mechanisms are less well understood than the self‐incompatibility (SI) mechanisms plants use to reject pollen from close relatives. Previous studies have shown that some interspecific reproductive barriers (IRBs) are related to SI in the Solanaceae. For example, the pistil SI proteins S‐RNase and HT protein function in a pistil‐side IRB that causes rejection of pollen from self‐compatible (SC) red/orange‐fruited species in the tomato clade. However, S‐RNase‐independent IRBs also clearly contribute to rejecting pollen from these species. We investigated S‐RNase‐independent rejection of Solanum lycopersicum pollen by SC Solanum pennellii LA0716, SC. Solanum habrochaites LA0407, and SC Solanum arcanum LA2157, which lack functional S‐RNase expression. We found that all three accessions express HT proteins, which previously had been known to function only in conjunction with S‐RNase, and then used RNAi to test whether they also function in S‐RNase‐independent pollen rejection. Suppressing HT expression in SC S. pennellii LA0716 allows S. lycopersicum pollen tubes to penetrate farther into the pistil in HT suppressed plants, but not to reach the ovary. In contrast, suppressing HT expression in SC. Solanum habrochaites LA0407 and in SC S. arcanum LA2157 allows S. lycopersicum pollen tubes to penetrate to the ovary and produce hybrids that, otherwise, would be difficult to obtain. Thus, HT proteins are implicated in both S‐RNase‐dependent and S‐RNase‐independent pollen rejection. The results support the view that overall compatibility results from multiple pollen–pistil interactions with additive effects.