Overwintering of <Emphasis Type="Italic">Taphrina wiesneri</Emphasis> within cherry shoots monitored with species-specific PCR

Taphrina wiesneri, the pathogen of witches’ broom of cherry, is highly pathogenic to Cerasus × yedoensis, the most widely planted ornamental cherry species in Japan. For adequate control of this disease, it is necessary to understand the life history of T. wiesneri. However, sites inhabited by T. wiesneri within infected trees are little understood, except during flowering and leafing periods in spring. Therefore, we attempted to detect the location of T. wiesneri in shoots of witches’ broom before flowering and leafing in spring using PCR with a T. wiesneri-specific primer pair that was designed from 69 sequences in rDNA-internal transcribed spacer region of 32 Taphrina species. DNA extracted from symptomatic and asymptomatic C. × yedoensis sampled before leafing was amplified by PCR. T. wiesneri was detected in every bud and 5-mm stem segment of symptomatic shoots, except for one stem segment, and locally inside buds and the inner bark of stem segments. These results indicate that T. wiesneri overwinters inside symptomatic shoots. Fungal hyphae were observed with an epifluorescence microscope in intercellular spaces of young leaves in symptomatic buds but not in asymptomatic ones in thin sections stained with fluorescein-isothiocyanate-conjugated concanavalin A. This observation supports the results of PCR detection.     

Characterisation of sunflower root colonisation by <Emphasis Type="Italic">Phoma macdonaldii</Emphasis>

Phoma macdonaldii, the causal agent of black stem disease of sunflower (Helianthus annuus), also attacks roots and collars of the plants, resulting in early death. Totally resistant lines do not exist for infection of the aerial parts, but tolerant lines have been characterised. This paper presents a study on colonisation of a partially resistant and a susceptible sunflower line by P. macdonaldii. The fungus was transformed with a constitutively expressed reporter gene encoding the jellyfish green fluorescent protein via Agrobacterium tumefaciens, and colonisation of sunflower roots by this transformed strain was studied by various microscopy techniques including confocal and scanning electron microscopy. The results show that penetration of the fungus into the root occurred through natural fissures or through the epidermis and was similar in both lines. In contrast, the colonisation rate of the stele was reduced in the partially resistant line, and the morphology of the fungal hyphae was also affected. The effect on hyphal morphology was strongest in the stele, indicating a localised production of defence compounds in this line.     

Pharmacokinetic disposition of triclabendazole in cattle and sheep; discrimination of the order and the rate of the absorption process of its active metabolite triclabendazole sulfoxide

A comparative pharmacokinetic study was conducted to determine the order and the rate of absorption of triclabendazole (TCBZ) in cattle and sheep. A commercial suspension of TCBZ (Biofasiolex, Biogénesis S.A., Argentina) was administered at a dose rate of 10 mg/kg by the oral route to six Holstein female calves and six Corriedale female sheep. The plasma concentration profiles of the metabolites triclabendazole sulfoxide (TCBZ-SO) and triclabendazole sulfone (TCBZ-SO₂) were analysed by means of the non-compartmental method. The order of the absorption process of the active metabolite, TCBZ-SO, was determined by construction of curves of cumulative absorbed fraction of the drug by means of the Wagner-Nelson method. The appearance of TCBZ-SO in plasma of cattle and sheep resembles the entry of a constant quantity of drug into the organism per unit time. This is explained by the reservoir effect of the rumen, which acts as a biological slow-release system for TCBZ-SO and its precursor TCBZ to the posterior digestive tract where they are absorbed. The plasma concentration profiles of TCBZ-SO in both species were well described by a one-compartment open model with zero-order process of absorption and first-order process of elimination. The values of AUC_0-∞ and C _max of TCBZ-SO did not differ between species, while other kinetic parameters except for λ _z had higher values in calves than in sheep. In the case of TCBZ-SO₂, t _max was the only parameter that did not differ between species, while other kinetic parameters except for λ _z had higher values in calves than in sheep.     

A simple and efficient method for isolating small RNAs from different plant species

Background  

Small RNAs emerged over the last decade as key regulators in diverse biological processes in eukaryotic organisms. To identify and study small RNAs, good and efficient protocols are necessary to isolate them, which sometimes may be challenging due to the composition of specific tissues of certain plant species. Here we describe a simple and efficient method to isolate small RNAs from different plant species.     

Spatial structure of shell polychromatism in <Emphasis Type="Italic">Cepaea hortensis</Emphasis> in relation to a gradient of landscape fragmentation in Western France

Because of their highly polymorphic shell patterns, Cepaea land snails have been the subject of numerous studies in ecological genetics. Here, we investigated the spatial structure of polychromatism in Cepaea hortensis in agricultural landscapes with zones from low to high hedgerow densities. Our main purpose was to search for a relationship between landscape composition and spatial structuring of chromatism. Despite significant spatial heterogeneity in the three landscapes sampled, only the high hedgerow density landscape showed a significant spatial structuring of shell polymorphism. In order to understand this result, an investigation of daily movement patterns in relation to habitat form was carried out on a mark-release experiment under semi-artificial conditions. This experiment revealed a strong influence of a linear corridor on snail dispersal. In the field, spatial heterogeneity of shell polymorphism, related to the effects of genetic drift, was shaped by restricted dispersal in narrow corridors. In the more enclosed one, i.e. where hedgerow density was the highest, the significant spatial structure we detected involved a balance between local genetic drift and environmentally mediated gene flow. This isolation-by-distance pattern resulted from direct gene exchange through fields between neighbouring populations. When applying landscape distances based on hedgerow length, no significant spatial correlation with polychromatism was found. In the more fragmented sites, random genetic drift seemed to be the prevailing force and, at the scale of the whole sampled area, selective pressures potentially interfere with these genetic drift-dispersal events.     

Landscape configuration alters spatial arrangement of terrestrial-aquatic subsidies in headwater streams

Context

Freshwater ecosystems depend on surrounding terrestrial landscape for resources. Most important are terrestrial leaf litter subsidies, which differ depending on land use. We lack a good understanding of the variation of these inputs across spatial scales.

Objectives

We sought to determine: (1) the relative importance of local versus catchment-level forestation for benthic leaf litter biomass in streams, (2) how landscape configuration alters these relationships, and (3) how land use affects the quality and diversity of leaf litter subsidies.

Methods

We measured biomass and identity of benthic leaf litter in 121 reaches in 10 independent catchments seasonally over the course of a year. We assessed direct and indirect effects of forestation, reach position, and seasonality on leaf litter biomass using structural equation models, and assessed how leaf litter diversity varied with land use.

Results

In catchments with forested headwaters, the degree of forestation and reach position in the catchment influenced benthic leaf litter biomass indirectly through local reach-scale forestation. In catchments where forest was only located downstream, or with minimal forest, none of these factors influenced reach-level benthic leaf litter. Leaf litter diversity peaked in fall in all land use types, but was generally lowest in forested reaches.

Conclusions

Not only habitat amount, but its location relative to other habitats is important for ecosystem function in the context of cross-ecosystem material flows. Here, lack of upstream forest altered spatial patterns of leaf litter storage. Studies with high spatiotemporal resolution may further reveal effects of landscape configuration on other ecosystems.

     

Generation and characterisation of Tn5-tagged <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> mutants that overcome <Emphasis Type="Italic">Xa23</Emphasis>-mediated resistance to bacterial blight of rice

Xanthomonas oryzae pv. oryzae causes bacterial blight of rice. Xa23, a bacterial blight resistance gene identified originally in wild rice, Oryza rufipogon, is dominant and resistant to all X. oryzae pv. oryzae field isolates tested. The corresponding avirulence gene avrXa23 is unknown. Here we report the generation of a random insertion mutant library of X. oryzae pv. oryzae strain PXO99 using a Tn5-derived transposon tagging system, and identification of mutant strains that are virulent on CBB23, a near-isogenic rice line containing Xa23. A total of 24,192 Tn5 inserted clones was screened on CBB23 by leaf-cutting inoculation and at least eight of them caused lesions on CBB23 comparable to those on JG30, the susceptible recurrent parent of CBB23. Polymerase chain reaction and Southern blot analysis showed that all the eight mutants, designated as P99M1, P99M2, P99M3, P99M4, P99M5, P99M6, P99M7 and P99M8, have a single Tn5-insertion in their genomes. The flanking DNA sequences of the Tn5-insertion sites were isolated by PCR-walking and sequenced. Bioinformatic analysis of the flanking sequences, by aligning them with the whole genome sequences of X. oryzae pv. oryzae strains PXO99, KACC10331 and MAFF311018 through NCBI, revealed that the Tn5-insertions disrupted genes that encode TAL effector AvrBs3/PthA, ISXo1 transposase, Type II secretion system protein-like protein or outer membrane protein, glycogen synthase, cytochrome C5 and conserved hypothetical protein. Further identification of these mutants will facilitate the molecular cloning of avirulence gene avrXa23. The authors C.-L. Wang, A.-B. Xu contributed equally to this work; Y. Gao and Y.-L. Fan contributed equally to this work.     

Characteristics of nucleotides and amino acids in cDNA sequence of xyloglucan endotransglycosylase cloned from <Emphasis Type="Italic">Anthocephalus chinensis</Emphasis>

Xyloglucan endotransglycosylase is an identified relaxation factor with functions of easing and extending of plant cell walls.Its activities are directly related to plant growth and elongation of organisms.Anthocephalus chinensis is a tall and fast growing evergreen tree species.We cloned the full cDNA sequence of AcEXT genes which is abundantly expressed in the cambium of A.chinensis.The sequence analysis of nucleotides and amino acids revealed the presence of a 1396 bp full cDNA sequence,including a 960 bp complete open reading frame(ORF) encoding a 320 amino acid protein.The deduced amino acid sequence of AcXET was homologous to the other known XET proteins and contained the conserved EIDFE catalytic site which was specific to all the XETs.Our data should serve as a foundation for further insight into AcXET gene molecular mechanisms during wood formation and cell wall engineering of woody plants.     

The occurrence of postharvest gray-mold rot of sweet persimmon caused by <Emphasis Type="Italic">Botrytis cinerea</Emphasis> in Korea

In this study, we identified the causative agent of postharvest gray-mold rot in sweet persimmon fruit that were collected from Gangneung, Gangwon Province, Korea in October 2016. Symptoms included extensive growth of mycelia on post harvested fruit. The fungus was isolated from infected fruit and cultured on potato dextrose agar (PDA). For identification of the fungus, we examined morphology characteristics and rDNA sequencing analysis of the fungus and confirmed its pathogenicity according to Koch’s postulates. The results of morphological examinations, pathogenicity tests, 5.8S rDNA sequences of the internal transcribed spacer regions (ITS1 and ITS4) and the five nuclear protein-coding genes G3PDH, HSP60, RPB2, MS547 and TUB revealed that the causal agent of postharvest gray-mold rot on sweet persimmon fruit in Korea was Botrytis cinerea.     

Virulence traits of avian pathogenic (APEC) and fecal (AFEC) <Emphasis Type="Italic">E. coli</Emphasis> isolated from broiler chickens in Algeria

Avian pathogenic E. coli (APEC) is the etiologic agent of avian colibacillosis, the most common disease responsible for chicken morbidity in the world. Although multiple virulence-associated factors were identified, their prevalence in Algeria is still poorly known. In the present research, 92 avian pathogenic E. coli (APEC) isolates were recovered from broilers with clinical signs and lesions of colibacillosis. In addition, 32 E. coli isolates collected from feces of healthy birds (AFEC) were included for comparison. All isolates were investigated by PCR for the presence of a total of 11 virulence-associated genes described for avian pathogenic (iroN, ompT, hlyF, iss, iutA, and fimC) and diarrheagenic E. coli (eae, stx, elt/est, ipaH, and aggR). The sensitivity of 39 APEC isolates to 16 antibiotics was also determined using antimicrobial pretreated microplates. Here, we report that 98% of the examined isolates host at least one of the tested virulence factors. The most prevalent genes in APEC were iutA (90.6%), ompT (86.9%), and iss (85.8%); whereas, iutA (78.1%), fimC (78.1%), and iroN (68.7%) were the highest prevalent genes in AFEC. Our data showed that none of the AFEC isolates harbor any of the tested diarrheagenic genes. Moreover, only elt/est (5.4%), stx (2.1%), and ipaH (2.1%) genes were carried by APEC isolates. We further established that ceftazodime, ceftiofur, mequindox, amoxicillin/clavulanic acid, and meropenem were the most efficient antibiotics against the analyzed APEC isolates. Overall, our findings provide more insights about APEC and AFEC virulence potential in Algeria which could participate in the fight against colibacillosis.