Crown Tissue Research Articles

Crown tissue proportions and enamel thickness distribution in early Pleistocene Homo antecessor maxillary premolars (Atapuerca, Spain)

AbstractObjectivesBoth morphometric and proteomic studies have revealed the close relationship of Homo antecessor with Neanderthals and H. sapiens. Considering this relationship, we aim to characterize the Early Pleistocene Atapuerca‐Gran Dolina (TD6) maxillary premolars to test if their pattern of enamel thickness is shared with Neanderthals or H. sapiens.Materials and MethodsWe employed microcomputed tomography to estimate 2D and 3D tissue proportions in seven H. antecessor maxillary premolars, belonging to two individuals: H1 and H3, and compared them to a sample of extinct and extant Homo populations of African, Asian and European origin (n = 52).ResultsOur results reveal a different pattern of enamel thickness between the Atapuerca‐Gran Dolina two individuals. While TD6‐H1 possesses thin‐enameled crowns, with a clear affinity with Neanderthals, TD6‐H3 exhibits the thick pattern, a trait shared with the majority of fossil hominins and H. sapiens.DiscussionThis work provides new data on upper premolar enamel thickness in H. antecessor. By documenting both a thin and a thick pattern of enamel thickness in the TD6 sample, we warn about the taxonomic utility of this feature in the characterization of isolated remains. We suggest that the thin enamel condition would have emerged during the Early Pleistocene and it became the most frequent and typical condition in Neanderthals. Possible causes for the pattern observed in TD6 include sexual dimorphism or presence of two populations in the sample; however, population variability is the most plausible explanation with a character expression intermediate between those of Neanderthals and other members of the genus Homo. This interpretation is compatible with the phylogenetic position of H. antecessor close to the ancestor of Neanderthals and H. sapiens.

First Report of Pantoea ananatis Causing Crown Necrobiosis on Strawberry in China.

Strawberry (Fragaria × ananassa) is an economically important crop in Zhejiang, China. In the autumn of 2021, crown necrobiosis and angular leaf spot was observed in commercial strawberry fields (cultivar 'fenyu') in Cixi, Ningbo, Zhejiang, China (N30°9'55″, E121°21'13″). The disease incidence ranged from 5 to 8 % in the field, but could reach 50 to 60 % in some heavily affected plastic tunnels. In the affected field, this disease could reduce strawberry production by 50%. Early symptoms were water-soaked lesions around the vein of the abaxial leaves; subsequently, reddish-brown irregular spots and coalesced lesions developed. In humid conditions, a sticky bacterial ooze exuding from lesions was observed. Finally, the crown of the diseased plant was necrotized, and several pockets were observed inside the crown after dissection. To isolate the causal agent, the infected leaves and crown tissues from six different plants were surface-sterilized with 75% ethanol for 1 min, rinsed twice with sterile distilled water, cut into small pieces, and soaked in 5 ml of sterile distilled water for 20 min. The supernatant from the cut-up pieces was serially diluted and spread on nutrient agar medium. After 2 to 3 days at 28℃, several yellow colonies were grown on the medium. The colonies from five infected plants were gram-negative, anaerobic rods, yellow, viscous, and gloss, which are typical characteristics of Erwinia anana (Wells et al. 1986). To confirm the identity of the causal bacteria, PCR was conducted for six randomly selected colonies to amplify 16S rRNA (Monciardini et al. 2002), fusA, and gyrB (Stice et al. 2002). The amplicons were sequenced and blasted, and the results showed that the six colonies were identical. The 16S rRNA, fusA, gyrB sequences of the isolate CM3 were deposited in GenBank with accession number ON754076.1, OP587277, and OP587278; BLAST search showed 99.93% (1445 bp out of 1446 bp), 100% (746 bp out of 746 bp), 99.64% (1371 bp out of 1376 bp) similarity with strains of Pantoea ananatis (KT741001.1, MH015093.1 and CP066803.1 accessions, respectively). The resulting concatenated data set of 16S rRNA-fusA-gyrB was used to build a multilocus phylogenetic analysis (MLSA) by maximum likelihood criteria. The MLSA tree indicated that the isolate CM3 belonged to Pantoea ananatis. The isolate's identity was further confirmed by P. ananatis-specific primers pagyrB-F/R (Xiao et al. 2022). Thus, this isolate was designated as P. ananatis CM3. To fulfill Koch's postulates, two old leaves were broken off each of the ten 2-month-old strawberry (cultivar 'fenyu') plants to create wounds, each plants was sprayed with a cell suspension ofP. ananatis (107CFU/ml, 0.5 ml) on the stem base. Ten plants were sprayed with water to serve as a control. All plants were kept at 28/25°C (day/night) under a 12-h/12-h photoperiod. All plants were covered with transparent plastic bags to maintain humidity. After 48 h, the bags were removed. After 2 weeks, water-soaked lesions on some leaves were observed similar to those in the field . Three to five weeks after inoculation, the crown of the inoculated plants was necrotized, which was similar to the symptoms in the field. No symptoms were observed in the control plants. The experiment was repeated three times. The bacteria were successfully reisolated from the inoculated crown tissues and leaves and confirmed as CM3 according to the same methodologies used for the initial identification. Bacterial leaf blight in strawberry caused byPantoea ananatishas been reportedin Nova Scotia, Canada, and Egypt (Bajpai et al. 2019; Abdel-Gaied et al. 2022). To our knowledge, this is the first report of Pantoea ananatis causing crown necrobiosis on strawberry in China. This report provides a basis for further research on this disease and its management and control.

Selection of resistant genotypes of Quince (Cydonia oblonga Mill.) to root and crown rot caused by the pathogen, Phytophthora cactorum, in the lab condition

Phytophthora root and crown rot is a common disease of different hortcrops in rosaceae family including pomefruits and stonefruits. In order to characterize reaction of different quince genotypes to Phytophthora, infected root and crown tissues of apple and quince trees in addition to some soil around them were collected from the orchards and cultured on semi-selective media. Four oomycete isolates out of 20 samples were recovered from which Phytophthora isolates were identified as P. cactorum, using physiological and morphological criteria. Disease resistance evaluation of quince genotypes to P. cactorum was implemented using excised twigs of 24 genotypes in glass bottles containing carrot agar media overlaid with a fresh layer of P. cactorum. The pathogenic isolate of P. cactorum selected from a pathogenicity test on unripe pear fruits. After two weeks of preservation of bottles in dark condition in 25 °C in growth chamber, the extent of necrosis was measured using a digital calipers then it was divided by length of twig to get the necrosis proportion. The genotypes showed a statistically significant different reaction to the pathogen at 1% level. Comparison of means using Duncan multiple range test indicated that the genotype ASP2 with 82% of necrosis proportion was the most susceptible one and PH2 (released as cultivar Behta), Unk and Khosro genotypes with 8, 10 and 13 percent necrosis of twigs were the most resistant genotypes, respectively.

Comparative Analysis of Antioxidant Accumulation under Cold Acclimation, Deacclimation and Reacclimation in Winter Wheat.

Low temperature during cold acclimation (CA) leads to the accumulation of detrimental reactive oxygen species (ROS) in plant tissues, which are scavenged by antioxidants, such as ascorbate and glutathione. However, there is a lack of studies examining the dynamics of antioxidants throughout CA, deacclimation (DEA), and reacclimation (REA) in winter wheat. Six winter wheat genotypes were selected to assess the effect of CA, DEA, and REA on the concentrations of ascorbate and glutathione in leaf and crown tissues under two CA temperature treatments. Higher levels of total, reduced, and oxidised ascorbate were determined in leaves, whereas crowns accumulated higher concentrations of nicotinamide adenine dinucleotide (NAD+) after CA, DEA, and REA. Constant low temperature (CLT) during CA led to higher contents of ascorbate and glutathione in both tissues at all stages of acclimation, in comparison with prolonged higher low temperature (PHLT). The concentrations of antioxidants increased after CA, tended to decrease after DEA, and returned to CA levels after REA. Significant positive correlations between freezing tolerance (FT) and antioxidants were only determined under the CA at CLT treatment, thus, affirming the negative effect of PHLT during CA on the FT of winter wheat.

Identification of Potential Zinc Deficiency Responsive Genes and Regulatory Pathways in Rice by Weighted Gene Co-expression Network Analysis

Zinc (Zn) malnutrition is a major public health issue. Genetic biofortification of Zn in rice grain can alleviate global Zn malnutrition. Therefore, elucidating the genetic mechanisms regulating Zn deprivation response in rice is essential to identify elite genes useful for breeding high grain Zn rice varieties. Here, a meta-analysis of previous RNA-Seq studies involving Zn deficient conditions was conducted using the weighted gene co-expression network analysis (WGCNA) and other in silico prediction tools to identify modules (denoting cluster of genes with related expression pattern) of co-expressed genes, modular genes which are conserved differentially expressed genes (DEGs) across independent RNA-Seq studies, and the molecular pathways of the conserved modular DEGs. WGCNA identified 16 modules of co-expressed genes. Twenty-eight and five modular DEGs were conserved in leaf and crown, and root tissues across two independent RNA-Seq studies. Functional enrichment analysis showed that 24 of the 28 conserved modular DEGs from leaf and crown tissues significantly up-regulated 2 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and 15 Gene Ontology (GO) terms, including the substrate- specific transmembrane transporter and the small molecule metabolic process. Further, the well-studied transcription factors (OsWOX11 and OsbHLH120), protein kinase (OsCDPK20 and OsMPK17), and miRNAs (OSA-MIR397A and OSA-MIR397B) were predicted to target some of the identified conserved modular DEGs. Out of the 24 conserved and up-regulated modular DEGs, 19 were yet to be experimentally validated as Zn deficiency responsive genes. Findings from this study provide a comprehensive insight on the molecular mechanisms of Zn deficiency response and may facilitate gene and pathway prioritization for improving Zn use efficiency and Zn biofortification in rice.

Metabolism of crown tissue is crucial for drought tolerance and recovery after stress cessation in Lolium/Festuca forage grasses.

A process of plant recovery after drought cessation is a complex trait which has not been fully recognized. The most important organ associated with this phenomenon in monocots, including forage grasses, is the crown tissue located between shoots and roots. The crown tissue is a meristematic crossroads for metabolites and other compounds between these two plant organs. Here, for the first time, we present a metabolomic and lipidomic study focused on the crown tissue under drought and recovery in forage grasses, important for agriculture in European temperate regions. The plant materials involve high (HDT) and low drought-tolerant (LDT) genotypes of Festuca arundinacea, and Lolium multiflorum/F. arundinacea introgression forms. The obtained results clearly demonstrated that remodeling patterns of the primary metabolome and lipidome in the crown under drought and recovery were different between HDT and LDT plants. Furthermore, HDT plants accumulated higher contents of primary metabolites under drought in the crown tissue, especially carbohydrates which could function as osmoprotectants and storage materials. On the other hand, LDT plants characterized by higher membranes damage under drought, simultaneously accumulated membrane phospholipids in the crown and possessed the capacity to recover their metabolic functions after stress cessation to the levels observed in HDT plants.

The effect of cold acclimation, deacclimation and reacclimation on metabolite profiles and freezing tolerance in winter wheat.

Global climate change will cause longer and warmer autumns, thus negatively affecting the quality of cold acclimation (CA) and reducing the freezing tolerance (FT) of winter wheat. Insufficient FT and fluctuating temperatures during winter can accelerate the deacclimation (DEA) process, whereas reacclimation (REA) is possible only while the vernalization requirement is unfulfilled. Six winter wheat genotypes with different winter hardiness profiles were used to evaluate the impact of constant low-temperature (2°C) and prolonged higher low-temperature (28 days at 10°C followed by 2°C until day 49) on shoot biomass and metabolite accumulation patterns in leaf and crown tissues throughout 49 days of CA, 7 days of DEA, and 14 days of REA. The FT of winter wheat was determined as LT30 values by conducting freezing tests after CA, DEA, and REA. Shoot biomass accumulation, projected as the green leaf area (GLA), was investigated by non-destructive RGB imaging-based phenotyping. Dynamics of carbohydrates, hexose phosphates, organic acids, proteins, and amino acids were assessed in leaf and crown tissues. Results revealed that exposure to higher low-temperature induced higher accumulation of shoot biomass and had a negative impact on FT of winter wheat. Prolonged higher low-temperature negatively affected the accumulation of soluble carbohydrates, protein content and amino acids, and had a positive effect on starch accumulation in leaf and crown tissues after CA, in comparison with the constant low-temperature treatment. DEA resulted in significantly reduced FT. Lower concentrations of glucose-6-phosphate, sucrose and proline, as well as higher concentrations of starch in leaves and crowns were found after DEA. The majority of the genotypes regained FT after REA; higher concentrations of glucose and malate in leaves, and sucrose in crown tissue were observed, whereas starch accumulation was decreased in both tissues. Negative correlations were determined between FT and starch concentration in leaves and crowns, while proline and proteins, accumulated in crowns, showed positive correlations with FT. This study broadens the knowledge regarding the effect of different low-temperature regimes on the dynamics of metabolite accumulation in winter wheat throughout CA, DEA, and REA, and its relationship to biomass accumulation and FT.

Comparison of Current Peanut Fungicides Against Athelia rolfsii Through a Laboratory Bioassay of Detached Plant Tissues.

Southern stem rot of peanut, caused by Athelia rolfsii, is an important fungal disease that impacts peanut production worldwide. Foliar-applied fungicides are used to manage the disease, and several fungicides have been recently registered for southern stem rot control in peanuts. This study compared fungicidal, residual, and potential systemic activity of current fungicides against A. rolfsii using a laboratory bioassay. Peanut plants grown in the field were treated with eight fungicides approximately 90 days after planting, and plants were collected for the laboratory bioassay weekly for 5 weeks following application. Peanut plants were separated into the newest fully mature leaf present at sample collection, the second newest fully mature leaf present at the time of fungicide application, the upper stem, and the crown tissues. Each plant tissue was inoculated with A. rolfsii then incubated at 30°C for 2 days. Lesion length was measured, and percent inhibition of fungal growth by each fungicide relative to the control was calculated. All fungicides provided the greatest inhibition of A. rolfsii on leaf tissues that were present at the time of fungicide application, followed by the newly grown leaf and upper stem. Little inhibition occurred on the crown. Fungal inhibition decreased at similar rates over time for all fungicides tested. Succinate dehydrogenase inhibitors provided less basipetal protection of upper stems than quinone outside inhibitor or demethylation inhibitor fungicides. Properties of the fungicides characterized in this study, including several newly registered products, are useful for developing fungicide application recommendations to maximize their efficacy in controlling both foliar and soilborne peanut diseases.

Prosthetic solution for whistling in fixed prostheses on implants: a case report

This clinical case report aims to describe the prosthetic solutions in a case of phonetic alteration reported by the patient after multiple anterosuperior rehabilitation with seven single cemented crowns type FP2, responsible for the lispting, reversing the prosthetic rehabilitation in four FP3 splinted and screwed crowns with palatal flange. Initially, the patient was unsatisfied with an upper removable partial denture. After the planning and execution of the surgical phase with eight osseointegrated implants, the osseointegration period occurred with the installation of seven single cemented crowns classified as FP2. During the adaptation period, the patient reported rattling during speech due to the passage of air in the cervical interdental region of the implant-supported prostheses (ISP), corresponding to teeth elements 22 to 12. When identifying the crowns responsible for the rattling, we elaborated a new plan with a FP3 splinted and screwed to guarantee reversibility, plus artificial cervical gum in the palatal region, called flange, replacing the lost anatomical crown and soft tissues. Several factors can result in phonetic changes; in the presented case, the multiple upper anterior prosthetic rehabilitation associated with the existence of a pronounced ogival palate and a thin gingival profile of the patient resulted in the development of whistling. The prosthetic conversion of crowns from FP2 to FP3 with palatal flange ensuring air locking between the cervical region of the FP proved satisfactory in resolving the condition of phonetic alteration, rehabilitating the lost phonemes.

Transcriptome analysis of Epichloë strains in tall fescue in response to drought stress

ABSTRACT Epichloë coenophiala, a systemic fungal symbiont (endophyte) of tall fescue (Lolium arundinaceum), has been documented to confer to this grass better persistence than plants lacking the endophyte, especially under stress conditions such as drought. The response, if any, of the endophyte to imposition of stress on the host plant has not been characterized previously. Therefore, we investigated effects on gene expression by E. coenophiala and a related endophyte when plant-endophyte symbiota were subjected to acute water-deficit stress. Plants harboring different endophyte strains were grown in sand in the greenhouse, then half were deprived of water for 48 h and the other half were watered controls. RNA was isolated from different plant tissues, and mRNA sequencing (RNA-seq) was conducted to identify genes that were differentially expressed comparing stress treatment with control. We compared two different plants harboring the common toxic E. coenophiala strain (CTE) and two non–ergot-alkaloid-producing Epichloë strains in tall fescue pseudostems, and in a second experiment we compared responses of E. coenophiala CTE in plant pseudostem and crown tissues. The endophytes responded to the stress with increased expression of genes involved in oxidative stress response, oxygen radical detoxification, C-compound carbohydrate metabolism, heat shock, and cellular transport pathways. The magnitude of fungal gene responses during stress varied among plant-endophyte symbiota. Responses in pseudostems and crowns involved some common pathways as well as some tissue-specific pathways. The fungal response to water-deficit stress involved gene expression changes in similar pathways that have been documented for plant stress responses, indicating that Epichloë spp. and their host plants either coordinate stress responses or separately activate similar stress response mechanisms that work together for mutual protection.

Genotypic, Temporal, and Tissue-type Effects on the Incidence of Tomato spotted wilt virus in Peanut (Arachis hypogaea L.)

Spotted wilt, caused by Tomato spotted wilt virus (TSWV), is one of the major diseases affecting peanut production in the southeastern United States. Growers use integrated disease management to control spotted wilt; however, disease resistance of a cultivar is the most important factor for managing this disease. This research evaluated genotypic, temporal, and tissue-type effects on the incidence of TSWV on each of four peanut cultivars. Four different types of tissues (leaf, root crown, old leaf and young leaf) were collected from each of four peanut cultivars (Florida-EPTM ‘113’, Florida-07, Georgia Green and Georgia Valencia) at five time points during the growing season. Immunoassays were conducted to detect viral presence. The incidence of TSWV infection increased over time during the growing season. Root crown tissue had higher infection than other types of tissue. Florida-EPTM ‘113’ had the lowest infection incidence. Compared to Florida-EPTM ‘113’, Florida-07 was seven times more likely to be infected by TSWV, while Georgia Green and Georgia Valencia possessed even higher probability, 18 times and 21 times higher, respectively. Florida-EPTM ‘113’ had significantly reduced TSWV infection likely due to slowing movement of the virus throughout the plant.

First Report of Crown Rot of Banana Caused by Fusarium proliferatum in Georgia, U.S.A.

Crown rot is one of the most damaging disease of banana fruit characterized by rot and necrosis of crown tissues. In severe cases, the disease can spread to the pedicel and banana pulp. Crown rot can be infected by several common fungi, including Lasiodiplodia theobromae, Musicillium theobromae, Colletotrichum musae, and a complex of Fusarium spp. and lead to softening and blackening of tissues (Lassois et al., 2010; Kamel et al., 2016; Triest et al., 2016; Snowdon, 1990). In November 2020, typical crown rot of banana fruits (cv. Pisang Awak, belonging to the tetraploid AABB genome) were observed from UGA Banana Research 12 Plots, Tifton, GA, with incidence rates of 15%. Initial symptoms appeared in the infected crown of green banana fruits. As the infection progressed, the crown tissues became blackened and softened, followed by an internal development of infection affecting the peduncle and the fruit, triggered early ripening of bananas. At last, the development of necrosis on the pedicels and fruits appeared and caused the fingers to fall off. To identify the pathogen, tissue pieces (~0.25 cm2) from the infected crown and pedicles were surface-sterilized in a 10% bleach solution for 1 min, followed by 30 s in 70% EtOH. The disinfected tissues were rinsed in sterile water 3 times and cultured on potato dextrose agar (PDA) amended with 50 µg/ml streptomycin at 25°C in the dark for 5-10 days. Isolates of the pathogen were purified using the single-spore isolation method (Leslie and Summerell 2006). Colonies on PDA produced fluffy aerial mycelium and developed an intense purple pigment when viewed from the underside. A range of colony pigmentation and growth rates were observed among the isolates. The microconidia were ovoid, hyaline, or ellipse in shape. The morphological features of the isolates were identified as Fusarium proliferatum (Leslie and Summerell, 2006). To further identify the isolates, genomic DNA was extracted from a representative isolate. And the internal transcribed spacer (ITS) region, the partial elongation factor (TEF1-α) gene and the β-tubulin gene (TUB2)were amplified and sequenced using the primers ITS1/ITS4 (Yin et al. 2012), EF-1 /EF-2 (O'Donnell et al. 1998) and B-tub1 /B-tub2 (O'Donnell and Cigelnik, 1997), respectively. The amplicons were sequenced and deposited in NCBI (accessions no. MZ292989, MZ293071 for ITS: MZ346602, MZ346603 for TEF1-α and MZ346600 and MZ346601 for B-tub). The ITS, TEF1-α, and B-tub sequences of the isolates showed 100% sequence similarity with Fusarium proliferatum isolates (accessions no. MT560212, LS42312, and LT575130, respectively) using BLASTn in Genbank. For pathogenicity testing, three whole bunched bananas sterilized with 10% bleach solutions and washed by sterilized water, were cut into 5 bananas per brunch. The cut surface of the banana crown was inoculated with conidial suspension (1.0 × 107 cfu/ml) of the pathogen with pipette tips. Equal number of bananas were treated with sterilized water in the same volume as a control. All bananas were sealed in a plastic bag and incubated at 25°C. After 7 days post inoculation, all inoculated bananas showed initial crown rot symptoms while no symptoms were observed on the control bananas. The fungus was re-isolated from the symptomatic tissues of infected bananas and confirmed to be genetically identical to F. proliferatum of the original inoculated strains according to morphological characteristics and molecular identification, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of F. proliferatum causing crown rot on bananas in Georgia, USA.

High-throughput sequencing data revealed genotype-specific changes evoked by heat stress in crown tissue of barley sdw1 near-isogenic lines

BackgroundHigh temperature shock is becoming increasingly common in our climate, affecting plant growth and productivity. The ability of a plant to survive stress is a complex phenomenon. One of the essential tissues for plant performance under various environmental stimuli is the crown. However, the molecular characterization of this region remains poorly investigated. Gibberellins play a fundamental role in whole-plant stature formation. This study identified plant stature modifications and crown-specific transcriptome re-modeling in gibberellin-deficient barley sdw1.a (BW827) and sdw1.d (BW828) mutants exposed to increased temperature.ResultsThe deletion around the sdw1 gene in BW827 was found to encompass at least 13 genes with primarily regulatory functions. A bigger genetic polymorphism of BW828 than of BW827 in relation to wild type was revealed. Transcriptome-wide sequencing (RNA-seq) revealed several differentially expressed genes involved in gibberellin metabolism and heat response located outside of introgression regions. It was found that HvGA20ox4, a paralogue of the HvGA20ox2 gene, was upregulated in BW828 relative to other genotypes, which manifested as basal internode elongation. The transcriptome response to elevated temperature differed in the crown of sdw1.a and sdw1.d mutants; it was most contrasting for HvHsf genes upregulated under elevated temperature in BW828, whereas those specific to BW827 were downregulated. In-depth examination of sdw1 mutants revealed also some differences in their phenotypes and physiology.ConclusionsWe concluded that despite the studied sdw1 mutants being genetically related, their heat response seemed to be genotype-specific and observed differences resulted from genetic background diversity rather than single gene mutation, multiple gene deletion, or allele-specific expression of the HvGA20ox2 gene. Differences in the expressional reaction of genes to heat in different sdw1 mutants, found to be independent of the polymorphism, could be further explained by in-depth studies of the regulatory factors acting in the studied system. Our findings are particularly important in genetic research area since molecular response of crown tissue has been marginally investigated, and can be useful for wide genetic research of crops since barley has become a model plant for them.

First Report of Neopestalotiopsis mesopotamica Causing Root and Crown Rot on Strawberry in Ecuador.

In Ecuador, strawberry production is located in the Andean region with an area of 1000 ha. Albion is the most popular cultivar due to its conical fruit shape, fruit size, bright red color, and sweetness. Since June 2014, farmers reported a reduction in the production cycle from 24 months to 6-8 months and a decreased yield of around 50% due to an unknown soil pathogen. Plant symptoms presented a reddish discoloration on new leaves, coming through the leaf apex to the petiole until turning wholly brown in old leaves leading to plant death. Additionaly, a brown-reddish spot inside the strawberry crown and root rot were reported (Fig. S1). In 2020, in Ecuador's most extensive production area, called Yaruqui (Pichincha province), 25 diseased plants were collected for pathogen isolation. The pathogen was isolated on water agar medium from the crowns internal tissue using 0.5 cm diseased plant fragments, previously disinfected with 2% sodium hypochlorite, and rinsed with sterile water. After two days, single hyphal tip was reisolated on potato dextro agar (PDA). A total of 18 pure isolates were grown at 25°C for 12 days, then 3-7 days of blacklight treatment was applied to induce sporulation. All the isolates presented a cottony beige mycelium with undulate edges. The conidia were ellipsoidal (range between 20.73 to 29 µm in length and 6.2 to 8.77 µm in width; n=60), multiseptated (4 segments) showing hyaline apical (3.8 to 5 µm) and basal (4.87 to 8 µm) cells, and three brown median cells, the second and third were darker than the fourth one, with one basal and 2 to 4 apical appendages (26.09 to 38.7 µm; Fig. S1). According to colony and conidia morphology, the isolates were identified as Neopestalotiopsis sp. (Dung et al. 2016; Essa et al. 2018; Maharachchikumbura et al. 2011). Five isolates were select randomly for DNA extraction and sequencing of the internal transcribed spacer (ITS) region (ITS4/ITS5), β-tubulin (Bt2b/ T1), and translation elongation factor 1-alpha (TEF-1a) region (EF1-728/ EF1-986) (Maharachchikumbura et al. 2014). DNA sequences obtained from each marker were identical for all isolates. Consensus sequences and alignment were built using ClustalX in MEGA X (Kumar et al. 2018). The consensus sequences were deposited in GenBank with the following accession numbers: ITS, MZ047602; β-tubulin, MZ054301; and TEF-1a, MZ054302. A multilocus Bayesian inference phylogenetic tree was constructed using the concatenated sequences in the Beast software (version 1.8.4)(Drummond et al. 2012; Maharachchikumbura et al. 2014). The isolate in our study clustered with isolates of Neopestalotiopsis mesopotamica with a posterior probability of 1, confirming its identity (Fig. S2). For Koch's postulates, healthy plants were grown in sterile soil for four months. Conidia of the pathogen were suspended in potato dextro broth (PDB) (1 x 104 conidia/ml), and it was sprayed on 15 healthy plants that previously had their crowns wounded with a sterile needle (0.6 cm deep) at the four cardinal points. The control treatment (15 plants) was wounded and sprayed with PDB alone. The plants were maintained at 25°C and more than 85% relative humidity (Sigillo et al. 2020). Twelve days after inoculation, plants showed reddish discoloration on new leaves, and old leaves presented low-level wilt, rusty color, and necrotic petioles. Forty-one days later, 75% of the treated plants had severe wilt or were dead, showing root and crown rot. Control plants presented no symptoms of the disease. Reisolation of the pathogen from the disease crown tissues was done on water agar and PDA as previously described. The isolates presented the exact morphology of pure cultures obtained from field diseased strawberry crowns. The pathogenicity test was performed twice. To our knowledge, this is the first report of Neopestalotiopsis mesopotamica being the causal agent of root and crown rot on strawberries in Ecuador. N. iranensis and N. mesopotamica have been reported as causal agents of strawberries fruit rot and leaf lesions in Iran (Ayoubi and Soleimani, 2016), and N. clavispora was reported to be causing root and crown rot on strawberry plants in Argentina (Obregon et al. 2018). Disease diagnosis contributes to providing strategies against this new disease. Further investigations are needed to find biological/chemical techniques or cultivar resistance to control this pathogen in strawberries in Ecuador.

Further analyses of the structural organization of Homo luzonensis teeth: Evolutionary implications

The species Homo luzonensis has recently been described based on a set of dental and postcranial elements found at Callao Cave (Northern Luzon, Philippines) and dated to at least 50–67 ka. Seven postcanine maxillary teeth are attributed to this taxon, five of them belonging to the same individual (CCH6) and representing the holotype of H. luzonensis, whereas the isolated upper premolar CCH8 and the upper third molar CCH9 are paratypes of the species. The teeth are characterized by their small dimensions associated with primitive features, as also found in Homo floresiensis, another hominin having evolved in an insular environment of Southeast Asia. Postcranial bones of the hands and feet of H. luzonensis and H. floresiensis show Homo habilis–like or australopith-like features, whereas cranial and dental morphology are more consistent with the Asian Homo erectus morphology. Due to this mosaic morphology, the origin and phylogenetic relationships of both H. luzonensis and H. floresiensis are still debated. To test the hypotheses that H. luzonensis derives from H. erectus or from an earlier small-brained hominin, we analyzed the µCT scans of the teeth. We investigated both external and internal tooth structure using morphometric methods including: crown outline shape, tooth crown tissue proportions, enamel-dentine junction shape, and pulp morphology. Homo luzonensis external crown morphology aligns more with H. erectus than with H. habilis/H. rudolfensis. The internal structural organization of H. luzonensis teeth exhibits more affinities with that of H. erectus and H. floresiensis than with Neanderthals and modern humans. Our results suggest that both H. floresiensis and H. luzonensis likely evolved from some H. erectus groups that dispersed in the various islands of this region and became isolated until endemic speciation events occurred at least twice during the Pleistocene in insular environments.

Growth and feeding ecology of coniform conodonts.

Conodonts were the first vertebrates to develop mineralized dental tools, known as elements. Recent research suggests that conodonts were macrophagous predators and/or scavengers but we do not know how this feeding habit emerged in the earliest coniform conodonts, since most studies focus on the derived, ‘complex’ conodonts. Previous modelling of element position and mechanical properties indicate they were capable of food processing. A direct test would be provided through evidence of in vivo element crown tissue damage or through in vivo incorporated chemical proxies for a shift in their trophic position during ontogeny. Here we focus on coniform elements from two conodont taxa, the phylogenetically primitive Proconodontus muelleri Miller, 1969 from the late Cambrian and the more derived Panderodus equicostatus Rhodes, 1954 from the Silurian. Proposing that this extremely small sample is, however, representative for these taxa, we aim to describe in detail the growth of an element from each of these taxa in order to the test the following hypotheses: (1) Panderodus and Proconodontus processed hard food, which led to damage of their elements consistent with prey capture function; and (2) both genera shifted towards higher trophic levels during ontogeny. We employed backscatter electron (BSE) imaging, energy-dispersive X-ray spectroscopy (EDX) and synchrotron radiation X-ray tomographic microscopy (SRXTM) to identify growth increments, wear and damage surfaces, and the Sr/Ca ratio in bioapatite as a proxy for the trophic position. Using these data, we can identify whether they exhibit determinate or indeterminate growth and whether both species followed linear or allometric growth dynamics. Growth increments (27 in Pa. equicostatus and 58 in Pr. muelleri) were formed in bundles of 4–7 increments in Pa. equicostatus and 7–9 in Pr. muelleri. We interpret the bundles as analogous to Retzius periodicity in vertebrate teeth. Based on applied optimal resource allocation models, internal periodicity might explain indeterminate growth in both species. They also allow us to interpret the almost linear growth of both individuals as an indicator that there was no size-dependent increase in mortality in the ecosystems where they lived e.g., as would be the case in the presence of larger predators. Our findings show that periodic growth was present in early conodonts and preceded tissue repair in response to wear and damage. We found no microwear and the Sr/Ca ratio, and therefore the trophic position, did not change substantially during the lifetimes of either individual. Trophic ecology of coniform conodonts differed from the predatory and/or scavenger lifestyle documented for “complex” conodonts. We propose that conodonts adapted their life histories to top-down controlled ecosystems during the Nekton Revolution.

Gene Expression in Cucurbita spp. Root and Crown during Phytophthora capsici Infection

Phytophtora capsici causes major diseases in cucurbit crops worldwide. In this study, we inoculated this pathogen into Cucurbita pepo subsp. pepo susceptible MUCU-16 and C. moschata tolerant M63. The gene expression of plant pathogenesis-related proteins chitinase (CpChiIV), lignin-forming peroxidase (CpLPOX), and defensin (CpDEF) and hormone-related enzymes salicylic acid (CpPAL) and ethylene (CpACO) was analyzed for two weeks post-inoculation in root and crown tissues. Differentially expressed genes were found between genotypes, tissues, days post-inoculation, and inoculated/non-inoculated samples. After inoculation, CpPAL and CpChiIV (crown) were downregulated in MUCU-16, while CpLPOX and CpDEF were upregulated in M63. In inoculated samples, higher expression changes were presented on days 10–14 than on day 3 for CpACO, CpLPOX, and CpDEF genes. Overexpression was higher for CpDEF compared to the other tested genes, indicating good suitability as a marker of biotic stress. The overexpression of CpDEF was higher in crown than in roots for both inoculated genotypes. The basal expression of CpPAL and CpDEF was higher in MUCU-16, but after inoculation, CpPAL and CpDEF gene expression were higher in M63. These changes suggest an association between CpDEF upregulation and tolerance, and between CpPAL downregulation and susceptibility.

Temporal expression study of miRNAs in the crown tissues of winter wheat grown under natural growth conditions

BackgroundWinter wheat requires prolonged exposure to low temperature to initiate flowering (vernalization). Shoot apical meristem of the crown is the site of cold perception, which produces leaf primordia during vegetative growth before developing into floral primordia at the initiation of the reproductive phase. Although many essential genes for winter wheat cold acclimation and floral initiation have been revealed, the importance of microRNA (miRNA) meditated post-transcriptional regulation in crowns is not well understood. To understand the potential roles of miRNAs in crown tissues, we performed a temporal expression study of miRNAs in crown tissues at the three-leaf stage, winter dormancy stage, spring green-up stage, and jointing stage of winter wheat grown under natural growth conditions.ResultsIn total, 348 miRNAs belonging to 298 miRNA families, were identified in wheat crown tissues. Among them, 92 differentially expressed miRNAs (DEMs) were found to be significantly regulated from the three-leaf stage to the jointing stage. Most of these DEMs were highly expressed at the three-leaf stage and winter dormancy stage, and then declined in later stages. Six DEMs, including miR156a-5p were markedly induced during the winter dormancy stage. Eleven DEMs, including miR159a.1, miR390a-5p, miR393-5p, miR160a-5p, and miR1436, were highly expressed at the green-up stage. Twelve DEMs, such as miR172a-5p, miR394a, miR319b-3p, and miR9676-5p were highly induced at the jointing stage. Moreover, 14 novel target genes of nine wheat or Pooideae-specific miRNAs were verified using RLM-5′ RACE assay. Notably, six mTERFs and two Rf1 genes, which are associated with mitochondrial gene expression, were confirmed as targets of three wheat-specific miRNAs.ConclusionsThe present study not only confirmed the known miRNAs associated with phase transition and floral development, but also identified a number of wheat or Pooideae-specific miRNAs critical for winter wheat cold acclimation and floral development. Most importantly, this study provided experimental evidence that miRNA could regulate mitochondrial gene expression by targeting mTERF and Rf1 genes. Our study provides valuable information for further exploration of the mechanism of miRNA mediated post-transcriptional regulation during winter wheat vernalization and inflorescent initiation.

Identification of Fungal Species Associated with Crown and Root Rots of Wheat and Evaluation of Plant Reactions to the Pathogens in North Dakota

Common root rot (CRR) and crown rot (CR), caused by Bipolaris sorokiniana and Fusarium species, respectively, can cause significant yield losses in cereal crops. To assess the prevalence, incidence, and severity of these diseases in North Dakota, wheat samples were collected from spring wheat fields across the state in 2012, 2013, and 2014. Based on sub-crown internode symptoms, a greater incidence and severity of CRR was observed in 2012 (warm and dry year) than in 2013 and 2014. Also, the Northwestern Glaciated Plains and Northwestern Great Plains ecoregions showed greater CRR incidence and severity compared to the Northern Glaciated Plains and Lake Agassiz Plains ecoregions in the state. Bipolaris sorokiniana and Fusarium species including F. acuminatum, F. avenaceum, F. culmorum, F. graminearum, F. equiseti, F. pseudograminearum, F. oxysporum, F. redolens, F. sporotrichioides, and F. solani were isolated and identified from the root and crown tissues of the wheat samples. B. sorokiniana was isolated more frequently than other fungal species in all sampled years and ecoregions of North Dakota. F. acuminatum, F. avenaceum, F. culmorum, F. equiseti, F. graminearum, F. pseudograminearum, and F. redolens were pathogenic causing infections on seedlings of the two wheat genotypes (ND652 and Alsen), whereas isolates of F. oxysporum and F. solani were non-pathogenic and considered as secondary invaders associated with the root and crown rot diseases. Evaluation of some spring wheat genotypes for reactions to one B. sorokiniana isolate at seedling and adult plant stages, and one F. culmorum isolate at the seedling stage indicated that susceptibility to these pathogens varied among different wheat genotypes tested. This study provides useful information on fungal species associated with root and crown rots of wheat in North Dakota and on resistant/susceptible reactions of some spring wheat lines to the different fungal isolates evaluated.

Improving of Winter Cold Hardiness by Glycine Betaine in Strawberry

One of the most important problems of strawberry cultivation in temperate regions is winter cold injuries. Current study investigated impacts of foliar application of glycine betaine (GB) at 0, 0.5, 1, 2 and 4 mM concentrations on the cold hardiness of strawberry. The plants were divided into two groups: one group for evaluation of cold hardiness at temperatures of -6, -9, -12, -15 and -18 °C; and the other for study of some biochemical characteristics. Results showed that GB treatment increased soluble carbohydrate and proline concentrations in both leaf and crown tissues, total protein concentration in leaf, and relative water content in leaf as compared to those in control. Based on LT50 calculated from electrolyte leakage and tetrazolium staining test, the GB application increased cold hardiness in strawberry plant based on its concentration. The highest cold hardiness was found in the 2 mM GB concentration based on electrolyte leakage and tetrazolium staining tests at -13.3 and -15.3 °C. Meanwhile, the lowest values of cold hardiness were observed in the control treatments based on electrolyte leakage and tetrazolium staining tests at -10.2 and -11.0 °C. Significant correlations were found between soluble carbohydrate and proline concentrations in leaf and crown, and total protein concentration with LT50 calculated from electrolyte leakage and tetrazolium staining test. We conclude that application of 2 mM GBhas the capacity to increase the freezing tolerance of strawberry and could be used as a prophylactic tool to reduce winter cold injury.