Crown Tissue Research Articles

Etiological agents of crown rot of organic bananas in Dominican Republic

Crown rot is a postharvest disease with a great negative impact on banana fruit quality. The infections occur at harvest, but the symptoms appear after overseas transportation. Different fungal pathogens are involved in crown rot, varying according to farming area. In this study we focused on etiology of organic banana crown rot in the Dominican Republic, which is one of the leading exporters of organic bananas. Bananas from five organic farms and their corresponding packing stations located in the Valverde province were studied. Over a period of three years, 558 banana hands were collected and a total of 5000 fungal colonies were obtained from the crown tissues and 518 representative colonies were purified, characterized and identified using morphological and molecular methods. Fungi were found in all samples from field and from packing house and were distributed in 11 genera. The fungal community was dominated by Fusarium, the most frequent genus (55%) found in more than 80% of all samples. The genus was represented by nine species; Fusarium incarnatum 53%, the most frequent, followed by Fusarium verticillioides 12%, Fusarium sacchari 12%, Fusarium proliferatum 7%, and Fusarium solani 6%. The five least frequent species were Colletotrichum musae, 7% overall frequency and found in 13% of all samples; Lasiodiplodia theobromae and Lasiodiplodia pseudotheobromae, 4% and 1% overall frequency, respectively, both species found in 7% of all samples; Nigrospora sp. 11% overall frequency, Alternaria spp. 6% overall frequency, followed by Phoma spp., Pestalotiopsis sp., Curvularia spp., Microdochium sp. and some other species, known to be saprophytes, with frequency lower than 2%. The etiological agents of crown rot disease can be ranked based on their presence and pathogenicity as follows: Fusarium incarnatum, Colletotrichum musae, Fusarium verticillioides, Fusarium sacchari, and Lasiodiplodia theobromae.

Comparison of adaptation of crowns of different lithium disilicate glass-ceramic

To evaluate the adaptation of different lithium disilicate glass-ceramic crown and whether different fabrication processes affect the adaptation of crowns. Thirty epoxy dies crowns were divided into 3 groups. They were cemented to domestic lithium disilicate glass-ceramic crowns in Group A, to IPS e.max CAD crowns in Group B and to IPS e.max Press crowns in Group C respectively. All crowns were cut by cutting machine. A confocal laser scanning microscope was used to measure the gap between crown tissue surface and die. There were significant differences in the three groups of measurement points(P<0.05). Within the groups, specimens in group A showed in the lowest marginal fit([46.8±9.1] μm). Those in group B showed the lowest shoulder suitability([59.3±7.9] μm), axial plane([50.5±3.6] μm) and occlusal surface([87.6±11.6 μm]. Those in group C demonstrated the lowest axial plane angle[(84.4±10.1) μm]. In addition to the axial plane angle, the CAD/CAM system exhibited good accuracy of fit. The three groups of lithium disilicate glass-ceramic crowns appeared to have clinically acceptable fit.

Rapid Crown Root Development Confers Tolerance to Zinc Deficiency in Rice.

Zinc (Zn) deficiency is one of the leading nutrient disorders in rice (Oryza sativa). Many studies have identified Zn-efficient rice genotypes, but causal mechanisms for Zn deficiency tolerance remain poorly understood. Here, we report a detailed study of the impact of Zn deficiency on crown root development of rice genotypes, differing in their tolerance to this stress. Zn deficiency delayed crown root development and plant biomass accumulation in both Zn-efficient and inefficient genotypes, with the effects being much stronger in the latter. Zn-efficient genotypes had developed new crown roots as early as 3 days after transplanting (DAT) to a Zn deficient field and that was followed by a significant increase in total biomass by 7 DAT. Zn-inefficient genotypes developed few new crown roots and did not increase biomass during the first 7 days following transplanting. This correlated with Zn-efficient genotypes retranslocating a higher proportion of shoot-Zn to their roots, compared to Zn-inefficient genotypes. These latter genotypes were furthermore not efficient in utilizing the limited Zn for root development. Histological analyses indicated no anomalies in crown tissue of Zn-efficient or inefficient genotypes that would have suggested crown root emergence was impeded. We therefore conclude that the rate of crown root initiation was differentially affected by Zn deficiency between genotypes. Rapid crown root development, following transplanting, was identified as a main causative trait for tolerance to Zn deficiency and better Zn retranslocation from shoot to root was a key attribute of Zn-efficient genotypes.

First report of Phytophthora inundata causing olive tree decline in Turkey

Young olive trees showing wilting and trunk cankers were observed in an orchard in Antalya province, Turkey. The aerial symptoms associated with reddish brown cankers expanding from the root collar to the stem were leaf discolouration, defoliation, and twig dieback. A Phytophthora sp. was consistently isolated from necrotic root and crown tissues of symptomatic trees. The causal agent of the disease was identified as Phytophthora inundata by morphological characteristics and comparison of sequences of the Internal Transcribed Spacer (ITS) region. Pathogenicity of the isolates was tested by stem inoculation and soil infestation using olive saplings. At the end of the experiments, lesions developed on both stems and roots, and root weight reduction occurred. This is the first report of P. inundata causing disease of olive trees in Turkey.

Technological qualities of defoliated and topped sugar beet

It is well known that beet with crown tissue have a higher content of melassigenic nonsugar substances than correctly-topped beet. However, there is a lack of information on the impact of this change on processing. Hence, the objective of this study was to determine the effect of defoliated beet on the different processing steps. Laboratory-scale processing experiments were conducted with defoliated and manually-topped beet from five sites in 2010/11 to 2012/13. Beet brei, raw press juice and thin juice have been analyzed, the subsequent effects have been simulated with a specially developed model. The results show that the beet composition changes increasingly when including a larger proportion of the crown tissue. Consequently, cutting off the first millimeters of the top of the crown will remove the worst part of the beet. Defoliated beet have a poorer technological quality compared to topped beet with a significantly higher content of melassigenic compounds, in particular -aminoN and invert sugar, and a lower pol. sugar content, thus resulting in a lower purity. This effect is similar for all products, beet brei, raw press juice, and thin juice. In thin juice, the effective alkalinity is significantly reduced, while colour and lime salts’ content are increased. Because of the lower quality/purity of defoliated beet more beet will have to be processed to produce the same quantity of white sugar, so the processing campaign will become longer. In the simulation model a lower thick juice purity and a higher molasses purity have been assumed for defoliated beet, as based on juice analysis and technological expectations. The higher content of nonsugars will result in an increased mass flow through the sugar house which will negatively affect the molasses exhaustion. At the same white sugar production, the simulation model calculates higher costs for processing aids and energy, but on the other hand also higher revenues for the additional production of pellets and molasses when processing defoliated beet.

Epiretinal Proliferation Associated with Macular Hole and Intraoperative Perifoveal Crown Phenomenon.

PurposeTo discuss the unique morphology and origin of epiretinal proliferation associated with macular hole (EPMH) occasionally observed in full-thickness macular hole (FT-MH) or lamellar hole (LH) and to introduce the perifoveal crown phenomenon encountered when removing this unusual proliferative tissue.MethodsSixteen patients showing EPMH in spectral domain-optical coherence tomography were selected from 212 patients diagnosed with MH, LH, FT-MH, impending MH, macular pseudohole, or epiretinal membrane between January 2013 and December 2014. Of the 212 patients included for clinical analysis, 33, 23, 11, 7, and 190 exhibited LH, FT-MH, impending MH, macular pseudohole, and epiretinal membrane, respectively. We reviewed visual acuity, macular morphology, and clinical course. Surgical specimens were analyzed histologically.ResultsEPMH presented as an amorphous proliferation starting from the defective inner/outer segment (IS/OS) junction covering the inner macula surface. Among the 16 patients with EPMH, 11 underwent vitrectomy, and all exhibited the intraoperative perifoveal crown phenomenon. EPMH tissue was sampled in three patients, one of whom had more tissue removed than intended and showed delayed recovery in visual acuity. Despite hole closure, IS/OS junction integrity was not successfully restored in four of 11 patients. Five patients were followed-up without surgical intervention. Visual acuity slightly decreased in three patients and did not change in one patient, while the remaining patient was lost during follow-up. Among the three perifoveal crown tissues obtained, two were successfully analyzed histologically. Neither tissue showed positivity to synaptophysin or S-100 protein, but one showed positivity to cytokeratin protein immunohistochemical staining.ConclusionsEPMH exhibited a distinct but common configuration in spectral domain-optical coherence tomography. An epithelial proliferation origin is plausible based on its configuration and histological analysis. Perifoveal crown phenomenon was observed when removing EPMH during vitrectomy.

Analysis of seasonal expression levels of wheat fructan exohydrolase (FEH) genes regulating fructan metabolism involved in wintering ability

In northern regions, winter wheat (Triticum aestivum L.) accumulates fructan during cold-acclimation in autumn and freeze-acclimation in early winter. The content of fructan in wheat crown tissues is associated with both freezing tolerance and snow mold resistance, and expression levels of fructan synthesis genes in leaf and crown tissue are correlated with both changes and varietal differences in fructan accumulation levels of wheat during cold- and freeze-acclimation. Fructan hydrolysis activity has also been thought be involved in wintering ability of wheat. Since several kinds of gene homologs encoding fructan exohydrolase (FEH: EC. 3.2.1.153, 154) with different substrate specificities have recently been cloned from wheat, changes in transcript levels of wheat FEH genes in field-grown wheat cultivars from autumn to spring were analyzed to investigate regulation of seasonal changes in fructan content. The seasonal expression patterns of five genes encoding 1-FEH, 6-FEH (and Wfh-sm3), 6&1-FEH and 6-KEH (kestose exohydrolase) varied. Among the five genes, only seasonal changes in the expression of wfh-sm3, which codes an enzyme that is able to hydrolyze almost all components of fructan that has accumulated in hardened wheat tissues, were correlated with those changes in fructan contents. Moreover, the transcript levels of wfh-sm3 were low in snow mold-resistant cultivars that accumulate high levels of fructan. The transcript levels of 6-FEH increased with decrease in ambient temperatures and the levels decreased under snow. The analysis indicated that cooperative expression of 6-FEH and 1-FEH genes might be related to the seasonal changes and varietal difference in mono- and disaccharide contents. This study showed that the coordinated expression of FEH genes in wheat was related to the regulation of water-soluble carbohydrate accumulation from autumn to early winter and fructan consumption under snow cover as well as energy supply and that wheat FEHs also play an important role in the varietal difference in freezing tolerance and snow mold resistance. In particular, the expression of wfh-sm3 may regulate fructan metabolism associated with tolerance for wintering stresses.

Transcriptional Analysis of Resistance to Low Temperatures in Bermudagrass Crown Tissues.

Bermudagrass (Cynodon dactylon L pers.) is one of the most geographically adapted and utilized of the warm-season grasses. However, bermudagrass adaptation to the Northern USA is limited by freeze damage and winterkill. Our study provides the first large-scale analyses of gene expression in bermudagrass regenerative crown tissues during cold acclimation. We compared gene expression patterns in crown tissues from highly cold tolerant “MSU” and susceptible “Zebra” genotypes exposed to near-freezing temperatures. Suppressive subtractive hybridization was used to isolate putative cold responsive genes Approximately, 3845 transcript sequences enriched for cold acclimation were deposited in the GenBank. A total of 4589 ESTs (3184 unigenes) including 744 ESTs associated with the bermudagrass disease spring dead spot were printed on microarrays and hybridized with cold acclimated complementary Deoxyribonucleic acid (cDNA). A total of 587 differentially expressed unigenes were identified in this study. Of these only 97 (17%) showed significant NCBI matches. The overall expression pattern revealed 40% more down- than up-regulated genes, which was particularly enhanced in MSU compared to Zebra. Among the up-regulated genes 68% were uniquely expressed in MSU (36%) or Zebra (32%). Among the down-regulated genes 40% were unique to MSU, while only 15% to Zebra. Overall expression intensity was significantly higher in MSU than in Zebra (p value ≤ 0.001) and the overall number of genes expressed at 28 days was 2.7 fold greater than at 2 days. These changes in expression patterns reflect the strong genotypic and temporal response to cold temperatures. Additionally, differentially expressed genes from this study can be utilized for developing molecular markers in bermudagrass and other warm season grasses for enhancing cold hardiness.

Genes Upregulated in Winter Wheat (Triticum aestivum L.) during Mild Freezing and Subsequent Thawing Suggest Sequential Activation of Multiple Response Mechanisms.

Exposing fully cold-acclimated wheat plants to a mild freeze-thaw cycle of −3°C for 24h followed by +3°C for 24 or 48h results in dramatically improved tolerance of subsequent exposure to sub-freezing temperatures. Gene enrichment analysis of crown tissue from plants collected before or after the −3°C freeze or after thawing at +3°C for 24 or 48h revealed that many biological processes and molecular functions were activated during the freeze-thaw cycle in an increasing cascade of responses such that over 150 processes or functions were significantly enhanced by the end of the 48 h, post-freeze thaw. Nearly 2,000 individual genes were upregulated more than 2-fold over the 72 h course of freezing and thawing, but more than 70% of these genes were upregulated during only one of the time periods examined, suggesting a series of genes and gene functions were involved in activation of the processes that led to enhanced freezing tolerance. This series of functions appeared to include extensive cell signaling, activation of stress response mechanisms and the phenylpropanoid biosynthetic pathway, extensive modification of secondary metabolites, and physical restructuring of cell membranes. By identifying plant lines that are especially able to activate these multiple mechanisms it may be possible to develop lines with enhanced winterhardiness.

The barley lipid transfer protein, BLT101, enhances cold tolerance in wheat under cold stress

To understand the mechanism by which the barley plants tolerate overwintering, we analyzed the BLT101 gene expression in four different cultivars including two each of spring and winter barleys, where the BLT101 transcript level varied among the cultivars with a long-term cold acclimation. The BLT101 transcript was induced at a higher level in the crown tissues in response to low temperature treatment. The BLT101 transcript level correlated with those of the corresponding protein. BLT101-overexpressing transgenic wheat lines (BLT101ox’s) grew slowly and showed decreased expression of the genes induced by hormones (such as auxin and cytokinin), compared to non-transgenic (NT) plants. After a prolonged cold treatment, the BLT101ox leaves showed normal phenotypes, while the NT plants displayed blighted and withered leaves. A substances leakage test showed that the BLT101ox wheat retains enhanced freezing tolerance, compared to the NT plants. The transgenic lines lost less water than the NT plants, under a cold-stressed condition. Our findings suggest that tolerance to water stress and the retardation of growth in cold conditions, the essential features in overwintering barleys, involve BLT101.

Studies into the phenolic patterns of different tissues of pineapple (Ananas comosus [L.] Merr.) infructescence by HPLC-DAD-ESI-MS (n) and GC-MS analysis.

In a comprehensive study, more than 60 phenolic compounds were detected in methanolic extracts from different tissues of pineapple infructescence by high-performance liquid chromatography with diode array detection and electrospray ionisation multiple-stage mass spectrometry (HPLC-DAD-ESI-MS (n) ) as well as by gas chromatography-mass spectrometry (GC-MS). The analytical workflow combining both methods revealed numerous compounds assigned for the first time as pineapple constituents by their mass fragmentations. Pineapple crown tissue was characterised by depsides of p-coumaric and ferulic acid. In contrast, major phenolic compounds in pineapple pulp extracts were assigned to diverse S-p-coumaryl, S-coniferyl and S-sinapyl derivatives of glutathione, N-L-γ-glutamyl-L-cysteine and L-cysteine, which were also identified in the peel. The latter was additionally characterised by elevated concentrations of p-coumaric, ferulic and caffeic acid depsides and glycerides, respectively. Two peel-specific cyanidin hexosides were found. Elevated concentrations of isomeric N,N'-diferuloylspermidines may be a useful tool for the detection of fraudulent peel usage for pineapple juice production. Mass fragmentation pathways of characteristic pineapple constituents are proposed, and their putative biological functions are discussed.

Identifying Resistance to Crown Rot Caused by Colletotrichum gloeosporioides in Strawberry.

Resistance to Colletotrichum crown rot (CCR, caused by Colletotrichum gloeosporioides) among commercial strawberry cultivars is variable, and increasing host resistance is a goal of the strawberry breeding program at the University of Florida. Twenty-eight accessions of Fragaria virginiana and F. chiloensis, the progenitor species of the cultivated strawberry (F. × ananassa), were evaluated for CCR resistance on artificially inoculated plants in a field trial, along with cultivars and breeding selections. Accessions PI 612320, PI 612323, and PI 551736 and selections FL 10-128 and FL 10-129 had no mortality in either of two seasons. The most susceptible genotypes had mortality between 75 and 100%. In a separate growth-chamber evaluation, seven genotypes were inoculated by spraying a spore suspension onto the crown or by injecting the suspension directly into the crown tissues. Mortality was higher using the injection method but the ranking of the genotypes was similar for both methods, indicating that resistance is expressed within crown tissues. Among the seven genotypes, selections FL 10-128 and FL 10-129 had the lowest mortality regardless of inoculation method, illustrating that a high level of resistance can be found within the cultivated germplasm.

Methyl Jasmonate Strengthens Wheat Plants Against Root and Crown Rot Pathogen Fusarium culmorum Infection

Root and crown rot of wheat, caused by Fusarium culmorum (Fc), is a serious disease worldwide, particularly in Iran. Currently, the mechanisms underlying resistance to Fusarium-caused diseases are still unknown. Methyl jasmonate (MeJA) has been identified as a vital cellular regulator, and the effect of exogenous MeJA application during wheat-Fc interaction has not been studied previously at the molecular level. This study, using real-time quantitative PCR, was carried out to determine the expression of seven host defense-associated genes consisting of PR3, PR4, PR5, TaPERO, LOX, PAL and cytochrome P450 gene (CYP709C1) at 48, 72 and 96 h after Fc-inoculation (hai) and to investigate the relationship between the induced resistance by MeJA application and the gene expression patterns. The results showed that several genes were induced earlier (48 hai) and to higher levels in the resistant cv. Sumai3 than in susceptible cv. Falat. Many Fc-induced genes were also activated and induced by MeJA particularly during the later stages of infection; however, in contrast to defense-gene induction by the pathogen, there was not a general trend of higher induction in Sumai3 compared to Falat following this chemical treatment. The chemically induced protection significantly reduced the development of necrotic symptoms in both cultivars over a 3-week period after inoculation; however, this reduction was greater in Falat than in Sumai3 relative to the controls. Surprisingly, the expression of these genes was also expressed in crown tissue that had not yet been in contact with the fungus, signifying that a form of systemic response was taking place in this interaction. This is the first work reporting the effect of timing application of MeJA during Fc infection and comparing the defense-related gene expression between root and crown tissues of wheat genotypes. Moreover, soil-drench application of MeJA resulted in strong induction, verifying the success of this application method in systemically activating JA signaling for the first time. These results provided important intimations for designing strategies to curtail diseases caused by Fusarium.

First Report of Fusarium Root Rot of Stored Carrot Caused by Fusarium avenaceum in Serbia.

Carrot (Daucus carota L. subsp. sativus (Hoffm.) Thell., Apiaceae), a widely consumed antioxidant-rich plant, is among the major vegetable crops grown in Serbia, with average annual production of 65,400 tons on approximately 7,000 ha (4). In May 2013, a severe root rot was observed on approximately 20% of cold-stored carrot roots originating from Gospođinci, South Bačka District, Serbia. Symptoms included dry rot of the collar and crown as well as large, brown to dark brown, circular, sunken lesions on the stored roots. Frequently, abundant whitish mycelium was observed covering the surface of the colonized roots. To determine the causal agent, small pieces of infected tissue were surface-disinfested with 2% NaOCl without rinsing, air-dried, and placed on potato dextrose agar. Five single-spore isolates obtained from collar and crown tissue sections, as well as nine isolates from root sections, all formed abundant, cottony white to pale salmon fungal colonies with reddish orange pigment on the reverse surface of the agar medium when grown at 25°C under 12 h of fluorescent light per day. All recovered isolates formed numerous, three- to six-septate, hyaline, needle-like, straight to slightly curved, fusoid macroconidia (30 to 80 × 4 to 5.5 μm, average 58.3 × 4.9 μm, n = 100 spores) each with a tapering apical cell. Microconidia of all isolates were generally scarce, two- to four-septate, spindle-shaped, and 15 to 35 × 3 to 5 μm (average 21.3 × 4.2 μm). Chlamydospores were not observed. Based on these morphological characteristics, the pathogen was identified as Fusarium avenaceum (Fries) Saccardo (1). The pathogenicity on carrot was tested for isolate 19-14 by inoculating each of five carrot roots surface-disinfected with 2% NaOCl, by placing a mycelial plug into the surface of a wound created with a cork borer. Carrot roots inoculated with sterilized PDA plugs served as a negative control treatment. After 5 days of incubating the roots at 25°C, root rot symptoms identical to those observed on the source carrot plants developed on all inoculated roots, and the pathogen was re-isolated from each of these roots using the same procedure descibed above. There were no symptoms on the control roots. Morphological species identification was confirmed by sequencing the translation elongation factor (EF-1α) gene (2). Total DNA was extracted directly from fungal mycelium of isolate 19-14 with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany), and PCR amplification was performed with primer pair EF-1/EF-2 (2). Sequence analysis of the EF-1α gene revealed 100% nucleotide identity of isolate 19-14 (GenBank Accession No. KM102536) with the EF-1α sequences of two F. avenaceum isolates from Canada (KC999504 from rye and JX397864 from Triticum durum). To our knowledge, this is the first report of F. avenaceum causing collar, crown, and root rots of stored carrot in Serbia. Since F. avenaceum can produce several mycotoxins, including moniliformin, acuminatopyrone, and chrysogine (3), the presence of this pathogen on stored carrots could represent a significant constraint for carrot production in Serbia, for both direct yield losses and potential mycotoxin contamination.

Line differences in Cor/Lea and fructan biosynthesis-related gene transcript accumulation are related to distinct freezing tolerance levels in synthetic wheat hexaploids

In common wheat, cultivar differences in freezing tolerance are considered to be mainly due to allelic differences at two major loci controlling freezing tolerance. One of the two loci, Fr-2, is coincident with a cluster of genes encoding C-repeat binding factors (CBFs), which induce downstream Cor/Lea genes during cold acclimation. Here, we conducted microarray analysis to study comprehensive changes in gene expression profile under long-term low-temperature (LT) treatment and to identify other LT-responsive genes related to cold acclimation in leaves of seedlings and crown tissues of a synthetic hexaploid wheat line. The microarray analysis revealed marked up-regulation of a number of Cor/Lea genes and fructan biosynthesis-related genes under the long-term LT treatment. For validation of the microarray data, we selected four synthetic wheat lines that contain the A and B genomes from the tetraploid wheat cultivar Langdon and the diverse D genomes originating from different Aegilops tauschii accessions with distinct levels of freezing tolerance after cold acclimation. Quantitative RT-PCR showed increased transcript levels of the Cor/Lea, CBF, and fructan biosynthesis-related genes in more freezing-tolerant lines than in sensitive lines. After a 14-day LT treatment, a significant difference in fructan accumulation was observed among the four lines. Therefore, the fructan biosynthetic pathway is associated with cold acclimation in development of wheat freezing tolerance and is another pathway related to diversity in freezing tolerance, in addition to the CBF-mediated Cor/Lea expression pathway.

First Report of Strawberry Dieback Caused by Lasiodiplodia theobromae.

With a typical Mediterranean climate, Aydin is the third largest strawberry-producing province, responsible for 13% of the overall strawberry production in Turkey. Strawberries (Fragaria × ananassa Duchesne) are mainly grown in raised, plastic-mulched beds under tunnels and soil solarization is the most effectively used management practice to control soil-borne pathogens. During October 2011 and 2012, 2 months after planting, wilting and collapse of plants were observed on commercial strawberry (cv. strawberry Festival) fields in Sultanhisar town of Aydin Province. Eleven percent of the plants were wilted and died. Symptomatic plants exhibited blackened necrotic discoloration of roots and in the cross section of crowns. A fungus was consistently isolated from pieces of infected tissue cut aseptically from the crowns and placed on potato dextrose agar. Fungus produced white colonies and later turned olivaecious black with dense aerial mycelium after 4 to 5 days incubation at 27°C. Dark brown to black pycnidia that formed on 20- to 30-day-old pure cultures under daylight conditions produced abundant conidia that were two-celled, thick-walled, and oval shaped with longitudinal striations. Single spore isolates from 12 samples were obtained and stored for further identification. The average size of 300 conidia was 25.42 ± 2.12 × 12.87 ± 1.08 μm. The morphology of the fungus was similar to Lasiodiplodia theobromae (Pat.) Griff. & Maubl. (syn. Botryodiplodia theobromae Pat.). To confirm the identity of the isolates, the internal transcribed spacer (ITS) region of ribosomal DNA and the elongation factor 1-alpha gene were amplified with the universal ITS1/ITS4 and EF1-688F/EF1-1251R (1) primers, respectively. The amplicons from 12 isolates were commercially sequenced at Macrogen (Korea) and were deposited in GenBank under consecutive accession numbers KF910369 to KF910380 and KJ641536 to KJ641547. Sequence comparison and phylogenetic analysis revealed that all 12 isolates were closely related and belonged to L. theobromae. Pathogenicity tests were performed by the toothpick technique (2) under greenhouse conditions (28°C, 14/10-h day/night, 70% RH) on potted strawberry plants (cv. strawberry Festival). Toothpicks carrying fungal growth taken from 1-week-old corn meal agar cultures of the tested isolates was placed into the basal crown tissue of the plants by piercing about 5 mm depth. Six plants were inoculated for each isolate and six were treated with sterile toothpick for control. All inoculated plants developed wilting and dieback symptoms resembling those of naturally infected plants within 2 to 3 weeks of incubation. All plants inoculated with the tested isolates collapsed after 4 weeks and showed discoloration of internal crown tissue. Control plants did not exhibit any disease symptoms, and crown tissue was symptomless. L. theobromae was successfully re-isolated from lesions of all inoculated plants. L. theobromae has been reported to cause cankers and dieback in a wide range of hosts in tropical and subtropical regions of the world (3). To the best of our knowledge, this is the first report of L. theobromae causing dieback on strawberry plants.

First Report of Fusarium Wilt of Strawberry Caused by Fusarium oxysporum in Serbia.

Strawberry (Fragaria × ananassa Duch.) is the third most important berry crop in Serbia with average production ranging from 30,000 to 35,000 t on approximately 5,000 ha (2). In June 2013, symptoms of wilt and whole plant collapse were observed on approximately 25% plants growing in commercial strawberry crop of cv. Alba in the locality of Zablaće (Moravica district). Initial symptoms included leaf chlorosis and wilt, followed by withering and necrosis of older leaves and reduced fruit production, eventually leading to plant collapse and desiccations. Internal vascular tissues of the crown showed distinct brown reddish discoloration. Three small pieces of infected roots, petioles, or crown vascular tissues were surface disinfested with 2% NaOCl and placed on five potato dextrose agars (PDA) per sample. After 7 days incubation at 23°C under 12 h of fluorescent light, nine monoconidial isolates were obtained (1) forming colonies with light purple mycelia. Colonies produced numerous hyaline, oval to ellipsoid microconidia (5 to 15 × 2.5 to 4.5 μm, average 8.45 × 2.25 μm), 3 to 5 septate fusoid macroconidia with pedicellate bases (20 to 50 × 2.70 to 6 μm, average 32.35 × 3.25 μm from 100 measured) and chlamydospores. Morphological and growth features were similar to the descriptions of Fusarium oxysporum Schlechtend emend. Snyder & Hansen (1). Pathogenicity of one selected isolate (97-13) was tested by dipping for 15 min the roots of five plants of each cultivar: Alba, Arosa, Clery, and Roxana into a conidial suspension (1 × 106 conidia/ml) harvested from a 7-day-old culture on PDA. Control plants were dipped in sterile distilled water. The inoculated plants were transplanted into pots containing sterilized peat and maintained in the greenhouse at 25°C. Thirty to thirty-five days post-inoculation, all plants developed wilt symptoms and vascular discoloration of crown tissues from which F. oxysporum was successfully re-isolated using the same method as for isolation. No symptoms were observed on any of the control plants. Morphological identification was confirmed by amplification and sequencing of a portion of the translation elongation factor-1 alpha (EF-1α) gene. Total DNA was extracted directly from fungal mycelium with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and PCR amplification performed with primers EF-1/EF-2 (4). Sequence analysis of EF-1α region revealed that Serbian isolate 97-13 (GenBank Accession No. KJ647280) shared 99 to 100% identity with the F. oxysporum sequences in GenBank. To our knowledge, this is the first report of Fusarium wilt on strawberry in Serbia. The presence of a new and potentially harmful disease may represent a serious constraint for strawberry production in Serbia.

Composite Inlays as A Modern Way of Posterior Restorations in the Dental Arch

Abstract Modern restorative dentistry offers many methods of restoring qualitative cavities within the clinical crowns. Due to the concept of maintaining continuity of the dental arch and maintaining the correct plane of occlusion, for reconstruction of posterior teeth, there are especially recommended indirect restorations, which are made in the dental laboratory, i.e. crown inlays. The aim of the study was based on the literature to describe indications, classifications and advantages of indirect composite inlays. Additionally, factors affecting the durability of indirect composite inlays and the influence on stomatognathic system were noted. Authors concluded that composite inlays can be recommended restoration method, not only in situations of loss of clinical crown tissue, but also in situations where it is necessary as structural reinforcement, e.g., in the case of Cracked Tooth Syndrome or Molar Incisor Hypomineralization.

First Report of Fusarium Wilt in Blueberry (Vaccinium corymbosum) Caused by Fusarium oxysporum in China.

Blueberry (Vaccinium corymbosum) production is developing quickly in China with about 20,000 ha presently cultivated. In 2010 in Lin'an, Zhejiang Province, plants developed an apparently new disease of blueberry (cv. Duke) with symptoms consisting of wilting of foliage, stunting of plants, and reduced fruit yields. Internal vascular and cortical tissues of plant crowns showed a brown to orange discoloration. Approximately 3% of the plants in the commercial plantings were affected and eventually died after 50 to 60 days. Infected plant samples (stems and roots) collected from different fields were surface sterilized with 1.5% sodium hypochlorite for 2 min, rinsed in water, plated on 2% potato dextrose agar (PDA), and incubated at 25°C in the dark for 1 week. Single conidium cultures were consistently isolated and cultured on acidified PDA (APDA) for morphological characterization (1,2). Colonies were light with purple mycelia, and beige or orange reverse colony colors developed after 7 days incubation at 25°C. Colonies producing abundant microconidia and macroconidia. Microconidia were hyaline and oval-ellipsoid to cylindrical (3.9 to 9.6 × 1.1 to 3.4 μm). Macroconidia were 3 to 5 septate and fusoid-subulate with a pedicellate base (28.6 to 37.5 × 3.3 to 4.2 μm). Morphology and development of macroconidia and microconida were consistent with a description of Fusarium oxysporum Schltdl (1,2). The ribosomal internal transcribed spacers ITS1 and ITS2 of eight isolates were amplified using primers ITS1/ITS4 on DNA extracted from mycelium and nucleotide sequences showed 100% similarity to that of F. oxysporum. To confirm pathogenicity, 20 blueberry plants (cv. Duke) were inoculated by dipping the roots into a conidial suspension (107 conidia per ml) for 30 min. The inoculated plants were transplanted into pots containing sterilized peat and maintained at 25°C and 100% relative humidity in a growth chamber with a daily 12-h photoperiod of fluorescent light. The pathogenicity test was conducted twice. Within 40 days, all inoculated plants developed wilt symptoms similar to that observed in the field. No symptoms were observed on plants dipped into distilled water. The fungus was successfully re-isolated from crowns and roots cultured on APDA, exhibiting morphological characteristics identical to F. oxysporum (1,2), confirming Koch's postulates. To our knowledge, this is the first report of blueberry wilt caused by Fusarium.

Carbohydrate and Lipid Dynamics in Wheat Crown Tissue in Response to Mild Freeze–Thaw Treatments

ABSTRACTFreezing tolerance resulting from cold hardening is critical to survival of fall‐seeded winter wheat. Exposure of winter wheat plants to cycles of freeze–thaw at temperatures just below, and just above freezing results in incremental improvements of freezing tolerance. Changes in the concentrations of carbohydrates in the cellular fluids of wheat crowns, and of lipids extracted from wheat crown tissue, were quantified following exposure to 24 h freeze(s) at –3°C, with or without a following thaw of 24 h at 3°C. Concentrations of simple sugars, and sucrose and related carbohydrates, increased in the first 24 h of exposure to –3°C, and continued to increase whether the plants were then exposed to a further 24 h at –3°C or were exposed to 3°C for 24 h. The concentration of fructans decreased during the first 24 h of exposure to –3°C, and continued to decrease if the plants were exposed to 3°C for 24 h, but increased if the plants were exposed to a continuous –3°C for 48 h. The frequencies of occurrences of numerous lipid species changed with each 24 h temperature treatment, some consistent with the behavior of lipid signaling molecules. Carbohydrates and lipids in wheat crowns are actively restructured at subzero temperatures as the plants acquire greater freezing tolerance. Identifying plant lines especially able to effect this restructuring may enable the development of plant lines with improved freezing tolerance.