Pieces Of Tissue Research Articles

First report of Colletotrichum fioriniae causing anthracnose on pear fruit in Serbia.

European pear (Pyrus communis L.) is one of the most important fruits in Serbia, with a total production of 55,938 tonnes in 2021 (Statistical Office of the Republic of Serbia). In November 2021, pear fruits (cv. Santa Maria) with typical anthracnose symptoms were collected in one storage facility in the area of Smederevo, Serbia. The disease incidence was about 2%. A total of nine infected fruits had dark, circular, sunken necrotic lesions with concentric rings of orange conidial masses produced in black acervuli. The tissue pieces from the edges of lesions (3 to 5 mm2) were surface-sterilized in 1% sodium hypochlorite for 1 min, rinsed twice in sterilized distilled water, air-dried, and incubated on potato dextrose agar (PDA) at 25°C. One isolate/diseased fruit was obtained (nine in total). Colony morphology of all isolates on PDA was light gray to pinkish on the upper side, and pink to vinaceous on reverse after 7 days of incubation at 25°C in the dark. Average growth rate was 64 ± 2 mm. The conidia were hyaline, fusiform, aseptate, smooth-walled, with both ends acute, and measured 12.8 to 16.6 µm × 3.5 to 4.5 µm (n =100). Appressoria were single, smooth-walled, dark brown, clavate to irregular outline, 5.5 to 9.6 × 4.8 to 7.2 μm (n =20). These morphological characterstics are consistent with the description of Damm et al. (2012) for Colletotrichum fioriniae. To confirm this identification, genomic DNA of representative isolate SZ-21-36 was extracted and the ITS region of the rDNA, partial beta-tubulin gene (BenA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and partial actin gene (ACT) were amplified using the primers ITS1/ITS4, Bt2a/Bt2b, GDF1/GDR1, and ACT512F/ACT783R, respectively (Hassan et al. 2019). The nucleotide sequence of each locus has been deposited in GenBank under Accession Nos. ON171625 (ITS), ON186696 (BenA), ON186697 (GAPDH), and ON186698 (ACT). Multilocus phylogenetic analysis based on the above mentioned molecular markers placed Serbian isolate of C. fioriniae with other isolates of this species deposited in GenBank (bootstrap support of 100%). Pathogenicity test was conducted on symptomless, detached pear fruits (cv. Santa Maria). Five surface-sterilized fruits were wound-inoculated with 50 μl of a conidial suspension (1 × 106 conidia/ml). After 10 days of incubation in a plastic chamber (25°C, 90% relative humidity, and 12-h photoperiod), typical anthracnose lesions developed on inoculated fruits. Five control fruits inoculated with sterile distilled water remained asymptomatic. The experiment was repeated once. Fungal colonies reisolated from the lesions showed the same morphological features as the original isolate. Pathogen was not reisolated from the control fruits. C. fioriniae was previously reported on European pear fruits in Croatia (Ivić et al. 2013), France (Da Lio et al. 2017), China (Fu et el. 2019), and in the USA (Pavlović et al. 2019). To our knowledge, this is the first report of anthracnose of pear fruit caused by C. fioriniae in Serbia. Currently, the economic impact of anthracnose caused by this pathogen is minimal, but its presence decreases commercial value and quality of pear fruits, and can be a risk to the further spread of C. fioriniae to other plant species.

Concomitant squamous cell carcinoma and actinomycosis—An unique and rare occurrence

: Actinomycosis of the oral cavity is not common, though actinomyces species often occur as normal commensals in the oral cavity. They gain entry into deep tissue to cause infection and consequent tissue damage whenever there is damage to the oral mucosa by injury or infection or neoplastic process. The case being discussed here is of a male, aged 62 years, who presented with an ulcero-proliferative lesion over left buccal mucosa. A punch biopsy was carried out on the lesion. Histopathology laboratory received three gray-white tissue pieces. Histological examination of the tissue pieces revealed features of moderately to poorly differentiated squamous cell carcinoma. Few colonies of radiating filamentous structures reminiscent of colonies of actinomyces species were noted. The sections were further stained with relevant stains for filamentous bacteria such as Periodic Acid Schiff (PAS) stain which confirmed the colonies of Actinomyces spp. The case being submitted here is an unique and relatively rare finding of concomitant squamous cell carcinoma and oral actinomycosis with only few cases being reported in publish English medical literature.

Understanding Hydra Regeneration

Understanding Hydra Regeneration Charisios Tsiairis from the Friedrich Miescher Institute for Biomedical Research explores Hydra Regeneration through genetic programs. Hydras inhabit pristine springs throughout the world and are shaped as hollow tubes constructed by two epithelial layers. These Hydras have a natural ability to regrow a whole organism from a small piece of tissue, and the animal is named after a mythical serpent monster that regrew two heads for every head chopped off. Hydra research can help identify genetic programs that may be present in our own cells, and help people to understand the mechanism of common diseases, such as multiple cancer. This link seems to be present in some of the earliest multicellular species, testifying to the importance of mechanical signals for the proper behaviour of cells.

Germplasm cryopreservation in bulls: Effects of gonadal tissue type, cryoprotectant agent, and freezing-thawing rates on sperm quality parameters

Germplasm preservation is crucial for reproductive programs involving farm and endangered species. This study describes the effects of slow-uncontrolled cryopreservation protocols on bovine sperm associated with testicular or epididymal tissues. Samples from the testis or epididymis (cauda) were cut into ∼0.5 or 1 cm3 fragments and cryopreserved using Me2SO (Dimethyl Sulfoxide) or glycerol-based cryoprotectants. Sperm were collected from testicular or epididymal tissue before and after freezing-thawing (38 °C or 40 °C) and kept at room temperature (RT) or 4 °C during handling. The parameters studied were viability, membrane integrity (HOS), motility, acrosome integrity, chromatin, and morphology. Pre-freezing parameters were lower in testicular sperm than epididymal: HOS+ and DNA integrity (P < 0.05). Normal-% pre-freezing testicular sperm morphology was lower than epididymal (43.3 ± 1.8% vs. 65.3 ± 14.8%). All testicular RT-kept sperm parameters decreased post-freezing, except for acrosome integrity, which remained constant (P > 0.05). There were no differences in Me2SO-frozen tissue sizes (P > 0.05). All epididymal RT-kept sperm parameters dropped post-freezing except for the constant DNA integrity (P > 0.05). 4oC-kept sperm were fitter than those at RT (P < 0.05). 4oC-kept testicular sperm viability, DNA, and membrane integrities declined after 38 °C or 40 °C thawing (P < 0.05). Acrosome integrity and motility remained unchanged after freezing (P > 0.05). 4oC-kept epididymal sperm acrosome integrity, motility, and HOS+% severely dropped post-thawing (P < 0.05). Viability and DNA integrity were unchanged (38 °C vs. 40 °C; P > 0.05). Overall, post-freezing sperm morphology was unaffected (P > 0.05), but Dag defect was significantly lower in testicular samples (P < 0.05). Whole-epididymis parameters were maintained up to 24h at 4 °C (P > 0.05). In conclusion, testis-epididymis freezing protocols should use small tissue pieces, Me2SO-based cryoprotectants, and 4°C-kept samples to reduce sperm damage.

First Report of Leaf Spot Caused by Alternaria alternata on Ligustrum japonicum in China.

Ligustrum japonicum is a small evergreen tree belonging to the Oleaceae and widely grown in China as a landscape ornamental and medicinal plant (Oh et al. 2021). In April 2021, a leaf spot disease was observed on Ligustrum japonicum in the campus of Anhui Agricultural University (31°51'4″N; 117°14'54″E), in Hefei City, Anhui Province, China. Approximately 50% of L. japonicum infections showed symptoms of round to oval, brown to dark brown lesions surrounded by a yellow halo. Diseased leaves were collected to determine the caused pathogen. Nine tissue pieces from three symptomatic leaves were surface sterilized with 2% NaClO for 1-2 minutes, followed by 75% ethanol for 1 minute, and then rinsed with sterile water for three times. The tissues samples were plated on potato dextrose agar（PDA）medium and incubated at 28 °C for 3 days. Seven fungal isolates were obtained from the plated tissues; the fungal hyphae were initially white and finally gray brown with flocculent aerial mycelia. Conidia were solitary or in chains, with various shapes, mostly subglobose. The size was (12.0-30.0) μm long and (6.0～12.0) μm wide (n=20). The cultural and morphological characteristics of these isolates were similar to those of Alternaria alternata (Simmons et al. 2007; Garibaldi et al. 2020). For accurate identification, genomic DNA was extracted from the mycelia of representative isolate (JSNZ). The internal transcribed spacer (ITS), 18S nrDNA (SSU), translation elongation factor 1-alpha (Tef1-α) and endopolygalacturonase (endoPG) sequences were amplified with the primer pairs of ITS1/ITS4, NS1/NS4, EFI-728F/EFI-986R and EPG-specific/EPG-3b, respectively (Woudenberg et al, 2013). The sequences were deposited in GeneBank under accessions MZ360963 (ITS), MZ677478 (SSU), OK274117(Tef1-α) and OK513186 (endoPG). BLAST analysis of the sequences of ITS, SSU, Tef1-α and endoPG showed >99% identity with those of A. alternata MK108918(561/601bp), KX609765 (1030/1035bp), LC132712 (281/281bp) and MT185591(459/483bp), respectively. A neighbor-joining phylogenetic tree was generated based on the concatenated data from sequences of ITS, SSU and Tef1-α using MEGA5.1, which clustered the present isolate with A. alternata strain CBS916.96 with high bootstrap support (100%). Based on cultural characteristics and phylogenetic analysis, the current isolate associated with leaf spot of L. japonicum was identified as A. alternata. Pathogenicity test was performed on three healthy L. japonicum on campus. Three healthy leaves of each plant were wounded with one sterile needle and inoculated with a 5-mm-diameter mycelial plug using sterile PDA plugs as control. The inoculated plants were covered with plastic bags and sprayed with water every 24 hours to maintain a high temperature and humidity environment. The experiment was repeated three times. After 12-days of incubation, symptoms were apparent on pathogen-inoculated plants, while the control plants remained asymptomatic. A. alternata was reisolated from inoculated leaves and matched the morphological and molecular characteristics of the original isolates, thus fulfilling Koch's postulates. To the best of our knowledge, this is the first report of a leaf spot disease caused by A. alternata in L. japonicum in China. Its identification will establish a foundation for managing the disease in China.

Human iPSC-derived midbrain organoids functionally integrate into striatum circuits and restore motor function in a mouse model of Parkinson's disease.

Rationale: Parkinson's disease (PD) is a prevalent neurodegenerative disorder that is characterized by degeneration of dopaminergic neurons (DA) at the substantia nigra pas compacta (SNpc). Cell therapy has been proposed as a potential treatment option for PD, with the aim of replenishing the lost DA neurons and restoring motor function. Fetal ventral mesencephalon tissues (fVM) and stem cell-derived DA precursors cultured in 2-dimentional (2-D) culture conditions have shown promising therapeutic outcomes in animal models and clinical trials. Recently, human induced pluripotent stem cells (hiPSC)-derived human midbrain organoids (hMOs) cultured in 3-dimentional (3-D) culture conditions have emerged as a novel source of graft that combines the strengths of fVM tissues and 2-D DA cells. Methods: 3-D hMOs were induced from three distinct hiPSC lines. hMOs at various stages of differentiation were transplanted as tissue pieces into the striatum of naïve immunodeficient mouse brains, with the aim of identifying the most suitable stage of hMOs for cellular therapy. The hMOs at Day 15 were determined to be the most appropriate stage and were transplanted into a PD mouse model to assess cell survival, differentiation, and axonal innervation in vivo. Behavioral tests were conducted to evaluate functional restoration following hMO treatment and to compare the therapeutic effects between 2-D and 3-D cultures. Rabies virus were introduced to identify the host presynaptic input onto the transplanted cells. Results: hMOs showed a relatively homogeneous cell composition, mostly consisting of dopaminergic cells of midbrain lineage. Analysis conducted 12 weeks post-transplantation of day 15 hMOs revealed that 14.11% of the engrafted cells expressed TH+ and over 90% of these cells were co-labeled with GIRK2+, indicating the survival and maturation of A9 mDA neurons in the striatum of PD mice. Transplantation of hMOs led to a reversal of motor function and establishment of bidirectional connections with natural brain target regions, without any incidence of tumor formation or graft overgrowth. Conclusion: The findings of this study highlight the potential of hMOs as safe and efficacious donor graft sources for cell therapy to treat PD.

First Report of Rhizopus oryzae Causing Rhizome Soft Rot of Ovate-leaf Atractylodes in South Korea.

Ovate-leaf Atractylodes (Atractylodes ovata) (OLA) is cultivated across South Korea to fulfil domestic need as a herbal medicine. In June 2021, the rhizomes with soft rot were observed in a commercial farmer field in Mungyeong, South Korea with 25% disease incidence. Initially, watery, black and soft lesion were observed on the rhizomes. Later, abundant aerial white mycelia covered the entire rhizome resulting in rot and death. Affected rhizomes were washed thoroughly using tap water, surface sterilized with 0.5% NaOCl and rinsed with sterile distilled water. Small pieces (2 to 5 mm2) of inner diseased tissues were cut, placed on potato dextrose agar (PDA) and incubated on at 25°C in the dark. Twelve morphologically similar pure fungal isolates were obtained by hyphal tipping twice on fresh PDA. Rhizoid, sporangiophores, sporangium, and sporangiospores were observed. Rhizoid and sporangiophores were nonseptate. Sporangia were globose while columella were globose or subglobose. Sporangiospores were numerous, varied in shape and size, subglobose, or oval, and measured 9.8 ± 1.5 (6.1 to 13.5) × 7.8 ± 1.1 (5.7 to 10.1) μm (n = 50). Morphological characteristics of the isolates were analogous to Rhizopus oryzae (Kwon et al. 2012, 2015). Sequences of the internal transcribed spacer (ITS) region and translation elongation factor 1-alpha (EF-1α) gene were analyzed for molecular identification. The primer pairs ITS1F/ITS4 and MEF10/MEF4 were used to amplify ITS and EF-1α respectively (Abe et al. 2007) and sequenced (Macrogen, Inc., Seoul, Korea). The obtained sequences were lodged with accession numbers LC705530 to LC705537. The ITS and EF-1α sequences were ≥ 99.5 % and ≥ 98.5% homologous with Rhizopus oryzae (strain CBS 381.52), respectively. In the maximum likelihood phylogenetic tree based on combined ITS and EF-1α sequences, the isolates formed a distinct clade with R. oryzae references isolates. Pathogenicity tests were conducted on healthy rhizomes with isolate R4. A conidial suspension (1× 106 conidia/mL) was sprayed on the surface of fifteen surface-sterilized rhizomes while fifteen rhizomes received sterile distilled water as controls. Treated rhizomes were placed in a plastic box containing wet paper tissues and incubated at 25°C in the12/12 day/light cycle. The typical rhizomes rot symptoms as seen in the field were observed on all inoculated rhizomes after 21 days of inoculation. The noninoculated rhizomes had no obvious symptoms. The causal fungus (3 isolates) was reisolated from inoculated rhizomes and identified as R. oryzae based on morphology and EF-1α sequence. This experiment was replicated twice. To the best of our knowledge this is the first report of R. oryzae infection on OLA rhizomes in South Korea. Rhizopus oryzae contained a complex of heterothallic closely related species and has been reported to cause 'Rhizopus rot' on various plants including apple, banana, mulberry, sweet potato, and tomato (Gnanesh et al. 2020; Khokhar et al. 2019; Kwon et al. 2012, 2015; Wang et al. 2017). Rhizome soft rot poses significant threat to OLA cultivation, sustainable management practices need to be adapted to control rhizome soft rot of ovate-leaf atractylodes.

Fusarium oxysporum f. sp. vasinfectum Identified on Okra in South Carolina, United States

Fusarium oxysporum f. sp. vasinfectum, the causal agent of Fusarium wilt on okra, was recovered in August 2020 on a commercial farm in South Carolina from cultivar Clemson Spineless, an heirloom cultivar still widely grown throughout the southeastern United States. An isolate recovered from discolored vascular tissue in the stem of a diseased plant was identified as F. oxysporum f. sp. vasinfectum based on sequences of the translation elongation factor 1-alpha and beta-tubulin genes. Two inoculation techniques were compared, injecting microconidia into stems and drenching microconidia around the base of plants. Drenching was a significantly more effective inoculation technique than stem injection ( P &lt; 0.05), with 77% and 40% of inoculated plants showing vascular discoloration and 73% and 45% of inoculated plants yielding colonies of F. oxysporum when pieces of vascular tissue were cultured on Komada's medium, respectively. This study provides the first confirmation of the presence of F. oxysporum f. sp. vasinfectum on okra in South Carolina.

First Report of Alternaria tenuissima Causing Leaf spot on Luffa cylindrica in China.

In August 2020-2021, symptoms of leaf spot were observed in luffa (Luffa cylindrical) fields in Qingdao city, Shandong Province. In all the 10 fields investigated, leaf spot occurred. The incidence (% luffa plants with symptoms from a defined number of plants assessed) was 35 to 60%. Early symptoms of infected leaves were small and irregular chlorotic lesions which later became irregular brown spots. As the disease progressed, the lesions gradually spread from the edge to the center of leaves to the middle, and became dark brown. The enlarged spots coalesced and eventually led to the withering and death of the leaves. In order to isolate the pathogen, 30 symptomatic leaves were collected from different planting fields. Small pieces of leaf tissues (5×5 mm) were cut from the junction of healthy and diseased tissues, sanitized with 2% NaClO for 1 min, rinsed three times with sterile distilled water. The tissue samples were then placed on potato dextrose agar (PDA) amended with 50 mg/L streptomycin sulfate, and incubated at 28℃ for 5 days in the dark. Ten purified fungal isolates were obtained by single spore isolation method. Colonies of these fungal isolates on the PDA medium were initially grayish-white, and then turned olive green with abundant cotton-like aerial hyphae. On potato carrot agar (PCA) medium, these fungi produced light brown and solitary conidiophore with septum. Conidia were obclavate or ellipsoid, brown, with 1-5 transverse septa and 0-3 longitudinal septa, and measured 13.2 to 49.5 × 9.5 to 21.6 µm (n=50). The morphological characteristics of these isolates were consistent with that of Alternaria spp. (Simmons 2007). The representative isolate NEAU-SG-1 was selected for molecular identification. The internal transcribed spacer (ITS) region of ribosomal DNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-α gene (TEF), histone 3 (HIS3), and RNA polymerase II second largest subunit (RPB2) were amplified using primer pairs ITS1/ITS4 (White et al. 1990), gpd1/gpd2 (Berbee et al. 1999), EF1-728F/EF1-986R (Carbone and Kohn 1999), H3-1a/H3-1b (Glass and Donaldson 1995), and RPB2-5F2/fRPB2-7cR (Sung et al. 2007), respectively. Sequences of these genes of isolate NEAU-SG-1 were deposited into GenBank database with the accession numbers of OL307719, OL415166, OL415169, OL415167, and OL415168. BLAST analysis of these sequences showed 99-100% homology with sequence homology with Alternaria tenuissima strains (ITS, MH824269; GAPDH, MK683783; TEF, MN056178; HIS3, MH824371; RPB2, LC621694). To fulfill Koch's postulates, ten surface disinfected 30-day-old luffa seedlings were inoculated by spraying conidia suspension (106 conidia/ml) of isolate NEAU-SG-1. The other ten surface disinfected seedlings inoculated with sterile distilled water served as the control group. After inoculation, each plant was covered with plastic bags for three days and cultured in greenhouse at 25℃. One week later, leaves inoculated with conidia suspension were observed with the same symptoms as described above, while the leaves of the control group were asymptomatic. Pathogenicity test was repeated twice. The Alternaria isolates were successfully re-isolated from those infected leaves and identified using the morphological and molecular methods described above. A. tenuissima has a wide host range in the world, and is the pathogen of leaf spot of many crops (Ma et al. 2021). To our knowledge, this is the first report of A. tenuissima causing leaf spot on luffa in China. This report will provide basic information for the diagnosis and prevention and control strategies of luffa leaf spot.

First report of thousand cankers disease caused by the fungus Geosmithia morbida and its vector Pityophthorus juglandis on Juglans regia in France

Thousand cankers disease (TCD) of walnut trees is a disease complex involving the ascomycete fungus Geosmithia morbida and its insect vector, the walnut twig beetle Pityophthorus juglandis. The disease is named after the large number of cankers that appear on the tree, caused by the wounds made by P. juglandis when it feeds and builds galleries under the bark and the parasitic behaviour of the fungus growing in the tissue surrounding the galleries. Several Juglans species may be affected. High mortality of walnut trees caused by TCD has been observed in the USA since the mid-1990's, mainly on Juglans nigra planted in urban areas. In 2013, TCD was reported for the first time in Italy, in the region of Veneto, on a small number of Juglans nigra of different ages (Montecchio & Faccoli, 2014). As part of a national survey organised by the French plant protection organization, pheromone traps for P. juglandis have been installed in some urban areas where Juglans spp. are present. In August and September 2022, scolytids were trapped in parks in the cities of Bron and Lyon (region of Auvergne-Rhône-Alpes, 45°43'17.6“N 4°54'00.3”E, and 45°46'33.7“N 4°51'09.0”E respectively), and sent to the laboratory for identification. The morphological characters matched the features of Pityophthorus juglandis according to Bright (1981) (Fig. 1) and the identity was confirmed by sequence analysis of the mitochondrial cytochrome oxidase I gene. The barcode region was amplified using the primer cocktail from Germain et al. (2013). The 658-bp long amplicon was sequenced (GenBank Accession No. OP975730) and BLAST analysis showed 100% identity with P. juglandis (KF725084), confirming identification. Following this finding, an active survey was triggered on walnut trees around these traps, looking for symptoms of TCD. Foliar wilt, twig and branch diebacks, small brown bark cankers on the branch and trunk (Fig. 2), sometimes with a cracked appearance (Fig. 3), as well as exit holes were observed on several Juglans regia trees of different ages. Removal of the bark around the cankers disclosed a brown discolouration of the phloem and the presence of bark beetle galleries (Fig. 4) where some P. juglandis were collected. Sections of branch or bark showing cankers were sampled and sent to the laboratory for analysis. After surface sterilisation with 70% ethanol (v/v), pieces of diseased tissues around the necrotic margins of the cankers were cut and plated onto malt extract agar (MEA) medium supplemented with 100 ppm chloramphenicol, and incubated at 22°C in the dark. After five to seven days, whitish to beige and flat-growing colonies were observed. After subculturing on MEA in the same conditions, the culture was slow growing, lobate, plane, white to yellowish (Fig. 5). All the features observed matched the description of Geosmithia morbida in culture made by Kolařic et al. (2011), such as the roughened penicillate conidiophores bearing narrowly cylindrical to ellipsoid conidia, 4–5 μm long, sometimes organized in persistent chains. Total DNA was extracted from two cultures and tested positive in real-time PCR with the G. morbida-specific assay developed by Rizzo et al. (2020). In addition, the rDNA internal transcribed spacer region was amplified for the two cultures using ITS5 and ITS4 primers. The 584-bp long amplicon was sequenced (OP930845 and OP930846) and BLAST analysis showed 100% identity with G. morbida (FN434082). Since this first finding, the presence of both the insect and fungus was also confirmed on J. nigra in the same area. An intensive survey is underway to determine the extent of the disease. To our knowledge, this is the second report of P. juglandis and G. morbida in Europe and the first in France, where eradication measures have been put in place. Their potential spread may endanger the existence of walnut trees and the associated economy in the whole continent. The mycology research unit of the ANSES Plant Health Laboratory (LSV) is supported by a grant managed by the French National Research Agency (ANR) as part of the French government's “Investing for the Future” (PIA) programme (ANR-11-LABX-0002-01, Laboratory of Excellence-ARBRE). We would like to thank Massimo Faccoli (University of Padua) for exchanges on Pityphthorus juglandis identity and the situation of thousand cankers disease in Europe.

Leaf spot on Photinia × fraseri caused by Neopestalotiopsis asiatica in China.

Photinia × fraseri is a well-known evergreeen ornamental tree. Owing to its flower-like red leaves and its ability to tolerate stressful environments, P. fraseri is widely cultured as a fast-growing hedge in southern China. From JulytoSeptemberin2021, a disease with symptoms similar to leaf spot was extensively observed on P. fraseri in Daozhen county (28° 51 'N, 107° 57 'E), Zunyi, Guizhou province, China. About 500 plants were surveyed and the incidence of leaf spot on P. fraseri leaves was 35% to 70%, significantly reducing the ornamentalandeconomicvalue. The symptomatic leaves displayed irregular, watery dark brown lesions with black conidiomata in gray centers, and 10 symptomatic leaves were collected from 10 trees.After surface sterilization (0.5 min in 75% ethanol and 2 min in 3% NaOCl, washed three times with sterilized distilled water) (Fang 2007), small pieces of symptomatic leaf tissue (0.2 × 0.2 cm) were plated on potato dextrose agar (PDA) and incubated at 25°C for about 7 days. Three single-spore isolates, GZAAS21-0327, GZAAS21-0328 and GZAAS21-0329, were obtained, which were identical in morphology and molecular analysis. Therefore, the representative isolate GZAAS21-0328 was used for further study. The pathogenicity of GZAAS21-0328 was tested through a pot assay. Ten healthy plants were scratched with a sterilized needle on the leaves. Plants were inoculated by spraying a spore suspension (106spores mL-1) of GZAAS21-0328 onto leaves until runoff, and the control leaves sprayed with sterile water. The plants were maintained at 28°C with high relative humidity (95%) in a growth chamber. The pathogenicity test was carried out three times (Fang 2007). The symptoms developed on all inoculated leaves but not on the control leaves. The lesions were first visible 72 h after inoculation, and typical lesions similar to those observed on field plants appeared after 15 days. The same fungus was reisolated and identified based on the morphological characterization and molecular analyses (ITS, TUB and TEF) from the infected leaves but not from the noninoculated leaves. Results of pathogenicity experiments of isolated fungi fulfilled Koch's postulates. Fungal colonies on PDA were villiform, creamy-white and sparse aerial mycelium on the surface with black, gregarious conidiomata. The conidia were fusoid, ellipsoid, straight to slightly curved, 4-septate, septa darker than the rest of the cell, and 23.0 (21.0 to 27.0) × 6.0 (5.0 to 7.0) µm (n=50). The morphological features were consistent with the descriptions ofNeopestalotiopsis asiatica Maharachch. & K.D. Hyde (Maharachchikumbura et al. 2012; Maharachchikumbura et al. 2014; Farr et al. 2022). The pathogen was confirmed to beN. asiaticaby amplification and sequencing of the internal transcribed spacer region (ITS), the partial β-tubulin (TUB) and partial translation elongation factor 1-alpha (TEF) genes using primers ITS4/ITS5, T1/Bt-2b and EF1-728F/EF-2, respectively (Maharachchikumbura et al. 2014). The sequences of PCR products were deposited in GenBank with accession numbers OK563071 (ITS), OK584020 (TUB) and OK663023 (TEF). BLAST searches of the obtained sequences revealed 100% (482/482 nucleotides), 99.05% (419/421 nucleotides), and 99.33% (891/897 nucleotides) homology with those ofN. asiaticain GenBank (JX398983, JX399018 and JX399049, respectively). Phylogenetic analysis (MEGA 6.0) using the maximum likelihood method placed the isolate GZAAS21-0328 in a well-supported cluster withN. asiatica. The pathogen was thus identified asN. asiaticabased on the morphological characterization and molecular analyses. To our knowledge, this is the first report of leaf spot on P. fraseri caused by N. asiatica in China. This study provides valuable information for the identification and control of the leaf spot on Photinia × fraseri.

First Report of Pectobacterium brasiliense causing Soft Rot on Conophytum luiseae in China.

Conophytum luiseae is native to the Namaqualand region of Cape, South Africa. It is a lovely plant with many short succulent spines on ingot-shaped fleshy leaf surfaces, and a high-value ornamental plant in China. In August to October 2021, a serious soft rot disease on Conophytum luiseae plants was observed in four greenhouses at a horticultural farm in Songjiang District, Shanghai, China. 70% of Conophytum luiseae plants on this farm had severe rot symptoms. Initially, wilting and soft rot symptoms appeared on fleshy leaves, then progressed into browning and withering symptoms of all fleshy leaves. To isolate and identify the causal agent, small pieces of lesion tissues were sterilized by 75% ethanol for 30 s, and rinsed three times with sterile water. Later, the tissues were crushed in sterile 2.0 mL centrifuge tube with 100 μl of sterile water. The suspension was serially diluted and spread on Luria-Bertani agar (LB) medium. After incubation at 28°C for 48 h, the bacterial colonies were tiny and streaked on LB plate for purification. After purification, five independent representative colonies were used for further confirmation. Genomic DNA from the bacterial isolate was extracted and used as the template to amplify 16s rDNA with primers 27F/1492R (Ying et al. 2012) and the housekeeping genes, dnaX with primers dnaXF/ dnaXR (Slawiak et al. 2009), and leuS with primers leuSF/ leuSR (Portier et al. 2019), respectively, by a polymerase chain reaction (PCR). The 16S rRNA sequences of one bacterial isolate was deposited in GenBank (GenBank accession OM333246) and showed a 99% similarity to that of Pectobacterium brasiliense (syn. Pectobacterium carotovorum subsp. brasiliense, Pcb) strains HG1501090309 (KU997683), BC1(CP009769), KC08 (KY021029). The dnaX (OM320998) and leuS (OM321306) sequences showed high similarity (> 99%) to P. brasiliense sequences. To further validate this identification, Pcb-specific primers BR1f/L1r was used for PCR, and it produced a predicted amplicon of 322 bp expected for P. brasiliense (Duarte et al. 2004). All five isolates could be detected by BR1f/L1r primer. To fulfill Koch's postulates, five healthy Conophytum luiseae were inoculated by spraying bacterial inoculum (108 CFU/ml), meanwhile five additional healthy Conophytum luiseae were implemented with sterilized distilled water as a negative control. The plants were then kept at 70% humidity and 25ºC. Seven days after inoculation, the inoculated plants showed serious soft rot, while the control samples remained healthy. Bacteria were re-isolated from rot of inoculated tissues, and the isolates were identified as the original pathogen by the 16S rRNA gene sequences. P. Brasiliense has been reported to cause soft rot on diverse plant hosts, like sweet potato, radish, tobacco (Liu et al. 2019; Voronina et al., 2019; Wang et al., 2017). Best to our knowledge, this is the first report that P. Brasiliense causes soft rot on Conophytum luiseae in China.

First Report of Maize White Spot Disease Caused by Pantoea ananatis in China.

Maize (Zea mays L.) is one of the most important crops in China. Since 2020, a new leaf spot disease has occurred in southwest China in areas such as Yunnan, Sichuan and Hubei provinces. Typical symptoms appeared after tasseling. The spots are scattered on the leaf surface, round to oval in shape with diameter range 3-20 mm. Spots are initially water-soaked, gradually turning yellow or white. In 2021, diseased leaf samples with typical white spot were collected for pathogen isolation and identification in Qujing, Yunnan province. Four small pieces of leaf tissue (about 0.25 cm2 in area) were excised from the edge of the necrotic lesion of each plant, surface sterilized with 75% ethanol for 1 min, rinsed three times with sterile distilled water, and soaked in sterile distilled water for 5 min. The solution was plated on Luria Broth medium (LB) plate (Shin et al. 2022) . After incubation at 28°C for 24 h, round, smooth-edged yellow colonies appeared in the LB plate. The bacterium isolated was gram-negative, negative for oxidase, positive for peroxidase, indole, citrate (Wells et al. 1987). Three strains (PA21QJ01, PA21QJ02 and PA21QJ03) showed identical colony morphology. PA21QJ01 was used for further molecular analyses. DNA was extracted from fresh colonies cultured in LB(Shin et al. 2022), and the fragments at the 16S rDNA, gyrB and rpoB loci were amplified using primers 27F/1492R (Galkiewicz and Kellogg 2008), UP-1/UP-2r (Yamamoto and Harayama 1995) and rpoBCM81-F/rpoBCM32b-R (Brady et al. 2008), respectively. The sequences of fragments of 16S rDNA, gyrB and rpoB from isolate PA21QJ01 were was deposited in GenBank (accession number: OM184301.1, OM302500, OM302499). A search for homologous sequences using BLAST resulted in 99.9, 99.6 and 99.8% identity of 16S rDNA, gyrB and rpoB, respectively, with sequences from the NN08200 of Pantoea ananatis (GenBank accession numbers: MK415050.1 for 16S rDNA, CP035034.1 for gyrB and CP035034.1.1 for rpoB). Above molecular results indicated that PA21QJ01 isolated from maize white spot is P. ananatis. Pathogenicity tests were performed on tasseled plants of the suscptible maize variety Wugu1790. After culture in LB medium plate at 30°C for 12 h, the bacterial solution was used for inoculation at a concentration of 1 × 108 CFU ml-1. After 7 days of inoculation, the leaves of the plants appeared water-soaked. After 10 days, white spot developed with brown margin. In contrast, the control plants remained healthy and symptomless. The same P. ananatis was reisolated in the inoculated maize plants, fulfilled Koch's law. In the last decade, P. ananatis has been reported to cause maize white spot in South Africa, Mexico, Poland, Argentina, Brazil (Sauer et al. 2015), and Ecuador (Toaza et al.2021). It has caused crop diseases with other crops, such as onion, rice, pineapple, melon, and sorghum, and others (Sauer et al. 2015). It caused leaf blight and leaf steak in rice in China (Yu et al. 2021). This is the first report of maize white spot caused by P. ananatis in China. However, to our knowledge, this is the first report of maize white spot disease in China. Attentions should be paid to screening for disease-resistant resources and breeding disease-resistant hybrids. Reference: Wells, J. M. et al. 1987. Int. J. Syst. Bacteriol. 37:136-143. Shin, G. Y. et al. 2022. Plant Dis. Doi: 10.1094/PDIS-08-21-1810-SC. Brady, C., et al. 2008. Syst. Appl. Microbiol. 31:447. Galkiewicz, J. P., and Kellogg, C. A. 2008. APPL ENVIRON MICROB, 74.24: 7828-7831. Toaza, A. et al. 2021. Plant Dis. Doi:10.1094/PDIS-02-21-0298-PDN Yamamoto, S., and Harayama, S. 1995. APPL ENVIRON MICROB, 61:1104.L. Sauer, A. V. et al. 2015. Agronomy Science and Biotechnology. Doi:10.33158/ASB.2015v1i1p21 Yu et al. 2021. Plant Dis. Doi:10.1094/PDIS-05-21-0988-PDN.

First Report of Epicoccum sorghinum Causing Leaf Spot on Paeonia suffruticosa in China.

Tree peony (Paeonia suffruticosa Andr.; Paeoniaceae) is highly valued in Chinese culture for their ornamental and medicinal benefits in anti-inflammation, anti-arrhythmia, activating the immune system and protecting the cardiovascular system (Zhai et al. 2020). In May from 2019 to 2021, leaf spot symptoms were observed on tree peony in an area of 0.8 hm2 in Bozhou, Anhui Province, China; with a disease incidence of 35%. Initially, black-brown spots (0.5-2.4 mm) appeared on the leaves, as the disease developed, the spots enlarged to form round, black lesions. Small pieces of tissue taken from the margins of lesions were surface-disinfected by 75% ethanol for 20 s followed by 0.1% mercuric chloride for 3 min, rinsed with distilled sterile water for three times, placed on potato dextrose agar (PDA) at 25 ºC for 7 days in the dark.Fifteen single spore isolates were obtained as described (Dong 2009). When cultured on PDA for 10 days, colonies were white, villose, then became gray-brown, with the reverse side becoming reddish-brown; Chlamydospores globose or irregular shaped, dark brown, unicellular or multicellular (4.3 to 12.2 × 13.1 to 33.7 µm) (n = 200). Pycnidia were black-brown, mostly spheroid, and 86 to 128 × 110 to 197 µm (n = 50); Conidia were hyaline, ellipsoidal, unicellular, aseptate, and 4.1 to 5.2 × 1.8 to 2.4 µm (n = 200) . The DNA of a representative isolate AF21-1-2 was extracted using the CTAB protocol (Doyle and Doyle 1987). The rDNA internal transcribed spacer (ITS), β-tubulin (TUB) and 28s nrDNA (LSU) genes were amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass et al. 1995), and LR5/LR0R (Vilgalys and Hester, 1990), respectively (OK485136, OK631973, and ON358331). BLASTn analysis showed that the ITS, TUB, and LSU sequences had 99% (497/501 bp), 98% (530/539 bp), and 99% (878/882 bp) identity with the Epicoccum sorghinum isolates ESCZO20 (MN944541), CZ01 (MT254851) and Dl16-338 (LN907481), respectively. Maximum likelihood phylogenetic tree revealed that AF21-1-2 clustered with E. sorghinum isolates. For pathogenicity test, leaves of 3-year-old healthy tree peony plant in the field were inoculated with 105 conidia/ml spore suspension (prepared with 30-day-old cultures on PDA), added with 0.1% (v/v) Tween-80. Ten surface-sterilized leaves per plant were sprayed with 30 ml suspension until runoff, a total of 3 plants were inoculated. Leaves sprayed with sterilized water serve as a negative control. Each plant was covered with a plastic bag to maintain high relative humidity at 15 to 23°C and was monitored daily for symptom development. Typical symptoms appeared on more than 80% of the leaves after 7 days of inoculation, while control leaves remained symptomless. Cultures isolated from inoculated leaves had the same morphological and molecular traits as those that were previously used to test Koch's postulates. Based on the morphological and molecular characteristics, the causal agent on tree peony was identified as E. sorghinum (Aveskamp et al. 2010). To our knowledge, this is the first report of E. sorghinum causing leaf spot on tree peony in China. The disease will cause serious economic loss for tree peony if it is not managed properly. This report may provide the basis for diagnosis and control of the disease.

First report of Diaporthe eres causing leaf spot on Platanus acerifolia in China.

Platanus acerifolia Willd. is one of the world famous urban greening trees, known as "the king of street trees" (Loretta et al. 2020). In August 2021, severe leaf spot disease was observed on P. acerifolia with 15% incidence on a street of Haidian district (116°29'E, 39°95'N), Beijing municipality, China. Typical symptoms were small and irregular brown spots with or without yellow haloes, which gradually expanded, coalesced and became necrotic lesions. For pathogen isolation, the leaf lesions were cut into small tissue pieces, disinfected by 0.3% sodium hypochlorite for 2 min and 70% ethanol for 40 s, rinsed in sterile distilled water, and then incubated on potato dextrose agar (PDA) plates. After incubation at 28°C for 4 days, three fungal isolates (FTDX2, FTDX3, and FTDX6) with similar colony characteristics were obtained after single spore isolation. Colonies were white with fluffy aerial mycelia, abundant pycnidia with black stomata appeared and cream-white liquid oozed after 20 days. Alpha conidia were 7.9 ± 0.6 × 2.5 ± 0.3 μm (n= 30), aseptate, hyaline, fusiform to ellipsoidal, and often biguttulate, while beta conidia were 22.7 ± 1.3 × 1.1 ± 0.1 μm (n= 30), aseptate, hyaline, linear, curved or hamate. The morphological characteristics were consistent with those of Diaporthe sp. (Udayanga et al. 2014). For further identification, total DNA was extracted from the three isolates. The internal transcribed spacer (ITS) region, translation elongation factor 1-α (EF1-α), beta-tubulin (TUB), calmodulin (CAL) and histone (HIS) genes were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), BT2a/BT2b (Glass and Donaldson 1995), CL1/CL2A (O'Donnell et al. 2000) and CYLH3F/H3-1b (Crous et al. 2014), respectively. The sequences were all deposited in GenBank (accession nos. OL870615 - OL870617 for ITS, OL870618 - OL870620 for EF1-α, OL870621 - OL870623 for TUB, OL870624 - OL870626 for CAL, and OL870627 - OL870629 for HIS) and aligned using BLASTn, obtaining 99-100% homology with the corresponding sequences of known Diaportheeresstrains in NCBI. Phylogenetic analysis of the combined sequences attributed the three isolates to the Diaportheeres clade. Pathogenicity tests were performed on three healthy one-year-old P. acerifolia plants using the randomly selected isolate FTDX2. The leaves were inoculated with 20 µl of spore suspension (106conidia/ml), with or without wound pretreatment, sterilized water inoculation under the same condition was used as control. All the treated plants were incubated in the greenhouse at 25°Cand 90% RH with a 12-h photoperiod. After 8 days,the inoculated plants showed spot symptoms on leaves similar to those previously observed, whereas the control leaves remained symptomless. Lesions on the wounded leaves were much larger in size compared with those unwounded. The same pathogen was re-isolated and identified based on morphological characteristics and gene sequencing data, fulfilling Koch's postulates. Diaporthe ereshas been reported to cause leaf spot on many horticultural plants, such as Photinia fraseri (Song et al. 2019) andPodocarpus macrophyllus (Zheng et al. 2020). To our knowledge, this is the first report ofD. erescausing leaf spot onPlatanus acerifoliain China. This finding is a valuable contribution to the knowledge on leaf spot disease development in horticultural plants.

Histopathological, ultrastructural, and immunohistochemical examination of changes in the placenta as a result of severe preeclampsia.

To investigate the role of hypoxia-inducible transcription factor-1 alpha (HIF-1α) and angiogenetic factor endothelin-1 (ET-1) expression in regulating hypoxia and placental development by routine histopathological methods. Twenty preeclamptic and normal placentas were used. Placenta tissue pieces were examined histopathologically after routine paraffin follow-ups. HIF-1α and ET-1 proteins were examined immunohistochemically, and placental tissues were examined ultrastructurally. Increase in syncytial proliferation, endothelial damage in vessels, and increase in collagen were observed in preeclamptic placentas. As a result of preeclampsia, an increase was observed in HIF-1α and ET-1 protein levels in the placenta. Dilatation of endoplasmic reticulum and loss of cristae in mitochondria were observed in trophoblast cells in preeclamptic placental sections. High regulation of oxygen resulting from preeclampsia has been shown to be a critical determinant of placentagenesis and plays an important role in placental differentiation, changes in maternal and fetal blood circulation, trophoblastic invasion, and syncytial node increase. It has been thought that preeclampsia affects secretion by disrupting the endoplasmic reticulum structure and induces mitochondrial damage, and that ET-1 may potentially help in the induction of stress pathways as a result of hypoxia in preeclampsia.

Ovaryum kriyoprezervasyonu ve yeniden transplantasyondan sonra Dnmt3a ve Dnmt3b protein ifadelerinin düşmesi bu süreçte DNA metilasyonunun bozulduğunu gösterir

Objective DNA methylation includes maintenance and de novo methylation process that are catalyzed by DNA methyltransferase (Dnmt) enzymes and these enzymes possess crucial roles in methylation of genes associated with the development of ovarian follicles. The aim of this study is to evaluate whether expression of Dnmt3a and Dnmt3b protein levels change after cryopreservation and re-transplantation of ovarian tissue in rats. Methods Four groups were designed as; fresh control (FC), frozen/thawed (FT), fresh re-transplanted (T), and frozen/thawed and re-transplanted (FTT). Slow freezing was used to cryopreserve the ovarian tissue pieces and expression of Dnmt3a and Dnmt3b proteins was assessed by immunohistochemistry. Result Expression of Dnmt3a and Dnmt3b significantly decreased in ovarian follicles in FT, T and FTT groups when compared FC. Conclusion Expression of Dnmt enzymes has been altered both after cryopreservation alone and following re-transplantation cycles which may lead to disturbed DNA methylation processes during rat folliculogenesis

Morphometrical and Histochemical study of glandular stomach (Proventriculus) in local domestic male ducks (Anas Platyrhynchos)

The present work explores morphometric and histochemical foundations of the wall of proventriculus in adult domestic male ducks. To achieve this study, six healthy adult ducks were purchased from local vendors in Mosul province. All birds were sedated. Their stomachs were removed after being dissected to achieve the gross description and measurements of the proventriculus, including the length and diameter of cranial, middle, and caudal regions of the proventriculus. Small tissue pieces 6-8 mm in length from three regions of proventriculus were processed for histomorphological studies using hematoxylin and Eosin stain, combined PAS-AB pH 2.5, toluidine blue, and Masson's trichrome stains. The finding showed that the proventriculus connects with the esophagus from the cranial side and the muscular stomach from the caudal side. It was a thin elongated oval tubular organ. Histologically, the mucosa of the proventriculus revealed the presence of folds and sulci-like structures covered by simple columnar epithelium. The tunica submucosa consisted of many signs of profound and straightforward sub-mucosal glands supported by connective tissue septa and extended from the basal part of the diverged gland through the lamina propria, which was organized in a conical manner (adenomere). Tunica Muscularis appeared as a double-layer coordinated in the form of inner longitudinal and outer circular ones. Finally, serosa consists of fibrous tissue, vessels, fatty tissue, and nerve plexuses studded by the mesothelial cells. The highest thickness of the four layers of mucosal, submucosal, muscular, and serosal layers in the distal part of ‎the proventriculus. Histochemical PAS staining positively reacted with epithelial tunica mucosa and wall of proventricular glands.

Associated pathogens to the regressive death of Canary palm (Phoenix canariensis) at urban areas of Queretaro, Mexico

The Canarian palm Phoenix canariensis palm has been severely affected during the past decade with high mortality of specimens of all ages in the metropolitan area of Queretaro, Mexico. The aim of this work was to determine the causal agent associated with the regressive death of P. canariensis in Santiago de Queretaro City. Therefore, inspections were made in order to find specimens with disease symptoms, and from the samples of rachis and apical meristem, pieces of tissue were left in wet chambers in order to promote of fungi and bacteria. A pathogenicity test from one isolate was done. A total of 262 palm trees were sampled: 95 without symptoms (36.3%), 122 death palms (46.6%) and 45 diseased (17.2%) with basal leaves death and flag leaf death. From 12 palms, Fusarium spp. (from the complex F. incarnatum and F. verticillioides) was molecularly identified. According to the morphological characteristics Erwinia sp. and F. solani were identified and with the last one pathogenicity test performed on twoyear-old palms. Also, the lethal yellowing disease was identified since an apical meristem sample was sent to the National Phytosanitary Reference Center. The results showed the presence of a complex of pathogens asscociated with the regressive death of P. canariensis.

Cytodifferentiation of fundic part of glandular stomach in non descript breed of Indian prenatal goat

Though the anatomy and physiology of the adult caprine (Capra hircus L.) stomach have been investigated extensively, the early development of the abomasum has not yet been fully elucidated. The glandular part of abomasum plays an important role in digestion of ingested food by action of gastric juices. Very few studies have been conducted so far regarding histogenesis of goat foetal abomasum in India. In the present study, we have investigated the embryonic and early foetal development of the goat, Capra hircus L. fundic abomasum. We collected 36 developing abomasum of healthy and normal embryos/foeti of goat and assigned them into three group viz. Gr. I (0-50 days of gestation), Gr. II (51-100 days of gestation) and Gr. III (101-150 days of gestation). Small pieces of tissues were processed by routine paraffin. The wall of glandular stomach, the fundic part, was composed of epithelium, pleuripotent blastemic tissue and serosa up to 44 days of gestation. Tunica muscularis became separable at 46 days of gestation. The epithelium was stratified type up to 50 days and gradually changed to pseudo-stratified columnar to simple columnar type from 76 days of gestation. Primary and secondary abomasal folds were observed at 51 and 76 days of gestation, respectively. Gastric pit, the fore runner of gastric gland was reported first at 70 days. The gland became branched tubular type at 145 days. The cells found in the mucosa of the abomasum were surface epithelial cells, chief cells, parietal cells, mucous neck cells and undifferentiated cells. Chief and parietal cell were observed at 76 days and mucous neck cells at 82 days of gestation. Reticular, collagen and elastic fibers came into sight at 38, 76 and 100 days of gestation, respectively. The present study is expected to supplement known data and knowledge regarding histogenesis of goat fetal abomasum and help in diagnosis and treatment of related congenital anomalies.