Small Pieces Of Tissue Research Articles

Leaf Spot Caused by Colletotrichum fructicola on Star Anise (Illicium verum) in China.

Star anise (Illicium verum) has been cultivated for centuries in southern China, and its fruit is an important seasoning spice, and can be used as a medicine (Wang et al. 2011). It is grown mainly in Guangxi, Guangdong, Guizhou, and Yunnan provinces, in China. Anthracnose is one of the important diseases of star anise, which seriously affects the yield and quality by infecting twigs, pedicels, fruit stalks and fruits (Liao et al. 2017). When leaf spots first appear, they are round, water-stained, small, dark brown spots, which expands into round separated spots, then the spots become yellowish brown with small black acervuli arranged in a circular pattern. On 22 August 2019, four leaf spot samples of star anise were collected, with two each from Shanglin County and Jinxiu County in Guangxi Province. The plantations in this area of around 8 ha had more than 80% leaf spot incidence. Small pieces of tissues (5 mm × 5 mm) were taken from the zone between symptomatic and healthy plant tissues, surface-disinfected in 75% ethanol for 10 s and 1% NaClO (sodium hypochlorite) for 1 min, and washed three times in sterilized distilled water. The sterilized leaf tissues were placed on potato dextrose agar (PDA) and incubated at 28°C in darkness for a week. Hyphae growing from tissue pieces were subcultured onto fresh PDA. Three of the four leaves yielded cultures resembling Colletotrichum spp. Four fungal isolates were obtained by a single-spore isolation method. The isolates JX1-2 and JX1-5 were collected from Jinxiu County while SL1-2 and SL2-1 were collected from Shanglin County. Genomic DNA was extracted from these four fungal isolates, followed by PCR amplification and sequencing of the rDNA internal transcribed spacer (ITS), actin (ACT), Apn2-Mat1-2 intergenic spacer, partial mating type (Mat1-2) (ApMat), calmodulin (CAL), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Weir et al. 2012). The sequences have been deposited in GenBank (ITS: MW301215 to MW301218; ACT: MW348965 to MW348968; ApMat: MW348973 to MW348976; CAL: MW348957 to MW348960; CHS-1: MW348969 to MW348972; GAPDH: MW348961 to MW348964). For phylogenetic analysis, MEGAX (Kumar et al. 2018) was used to produce a Maximum Likelihood (ML) tree with 1000 bootstrap replicates, based on a concatenation of the sequenced genomic regions for each of the four isolates from this study as well as sequences of other Colletotrichum species obtained from GenBank. The results revealed that isolates JX1-2, JX1-5, and SL1-2 were C. horii, and SL2-1 was C. fructicola (Weir et al.2012). The resulting colonies were initially white with abundant aerial hyphae, and white-gray after three days at 28°C on PDA. Isolate SL2-1 eventually turned greenish-grey after 14 days, while the center of C. horii isolates turned iron-gray with white-gray marginal. Both species of Colletotrichum had hyaline conidia that were terete, smooth, apex obtuse, base truncate, and there were no significant differences (P>0.05) in conidial size between C. horii (10.5 to 33.6 × 3.6 to 9.3 μm) (n=300) and C. fructicola (13.1 to 16.2 × 4.7 to 7.1 μm) (n=100). Pathogenicity tests were conducted in the greenhouse on 1-year-old star anise seedlings, and performed with a conidial suspension (10 µL of 106 conidia/mL) containing 0.1% Tween 20 placed onto lightly wounded sites on healthy leaves. Light cross-shaped wounds were made with sterilized toothpicks, gently scratching the surface without piercing the leaf. Each isolate was inoculated onto three seedlings, with at least eight leaves per seedling inoculated in two spots after light wounding. Control seedlings were inoculated with water containing 0.1% Tween 20. All inoculated seedlings were maintained in the greenhouse (12 h/12 h light/dark, 25±2°C), and covered with plastic bags to maintain high humidity throughout. The wounded sites inoculated with C. horii darkened to greenish-brown after 24 h, and C. fructicola gave similar symptoms after 36 h. Then the wounds turned to light brown round spots, and after 5 days, the spots expanded to water-stained spots with dots of acervuli arranged in a circular pattern. No symptoms were observed for the non-inoculated control. Each fungal isolate was consistently re-isolated from inoculated leaves, thus fulfilling Koch's postulates. There were significant differences (P<0.05) in aggressiveness between the two species, with C. horii showing larger diameter lesions (averaging 10.2 mm) than C. fructicola (averaging 8.4 mm). Anthracnose of star anise caused by C. horii (Liao et al. 2017) and C. coccdes (Wu et al. 2003) has been previously reported in China; however, to our knowledge, this is the first report of C. fructicola infecting star anise in China. This study may provide reference for further epidemiological study and prevention of anthracnose on star anise.

Testicular Torsion: Preliminary Results of In Vitro Cell Stimulation Using Chorionic Gonadotropin

Testicular torsion is a pathology that occurs in young males generally before the age of 25. Despite surgery representing the only effective approach, there is still a need to identify a marker that can predict whether a preserved testicle will be functional. In addition, no therapeutic approach is currently considered in the post-operative phase. Through an approach based on the in vitro culture of a tissue strictly linked to the testicle, the gubernaculum, we defined the healthy state of the organ and the possible responsiveness to a therapy used in the andrology field, chorionic gonadotropin (hCG). Firstly, we optimized a protocol to obtain viable cells starting from a small piece of gubernacular tissue harvested during surgery with the aim to amplify cells in vitro. Intriguingly, only for a patient whose testicle had been removed during surgery due to an excessive necrotic area, gubernacular cells were not able to grow in culture. These data support the possibility of exploiting the gubernaculum to evaluate the healthy state of the testicle. Then, as we demonstrate that gubernacular cells express a luteinizing hormone receptor, to which hCG is specific, we analyzed the cellular response to hCG treatment on in vitro cultured cells derived from patients affected by testicular torsion. Our study opens the way for the possibility of evaluating testicle wellbeing after derotation through in vitro culture of a small piece of gubernaculum together with predicting the response to the treatment with hCG, which can have a positive effect on cell proliferation and vascularization.

Towards a Comprehensive Optical Connectome at Single Synapse Resolution via Expansion Microscopy.

Mapping and determining the molecular identity of individual synapses is a crucial step towards the comprehensive reconstruction of neuronal circuits. Throughout the history of neuroscience, microscopy has been a key technology for mapping brain circuits. However, subdiffraction size and high density of synapses in brain tissue make this process extremely challenging. Electron microscopy (EM), with its nanoscale resolution, offers one approach to this challenge yet comes with many practical limitations, and to date has only been used in very small samples such as C. elegans, tadpole larvae, fruit fly brain, or very small pieces of mammalian brain tissue. Moreover, EM datasets require tedious data tracing. Light microscopy in combination with tissue expansion via physical magnification—known as expansion microscopy (ExM)—offers an alternative approach to this problem. ExM enables nanoscale imaging of large biological samples, which in combination with multicolor neuronal and synaptic labeling offers the unprecedented capability to trace and map entire neuronal circuits in fully automated mode. Recent advances in new methods for synaptic staining as well as new types of optical molecular probes with superior stability, specificity, and brightness provide new modalities for studying brain circuits. Here we review advanced methods and molecular probes for fluorescence staining of the synapses in the brain that are compatible with currently available expansion microscopy techniques. In particular, we will describe genetically encoded probes for synaptic labeling in mice, zebrafish, Drosophila fruit flies, and C. elegans, which enable the visualization of post-synaptic scaffolds and receptors, presynaptic terminals and vesicles, and even a snapshot of the synaptic activity itself. We will address current methods for applying these probes in ExM experiments, as well as appropriate vectors for the delivery of these molecular constructs. In addition, we offer experimental considerations and limitations for using each of these tools as well as our perspective on emerging tools.

First Report of Anthracnose Caused by Colletotrichum liaoningense on Trichosanthes kirilowii in China.

Trichosanthes kirilowii Maxim (T. kirilowii) is widely grown in central China for its medicinal and edible value. In August 2020, an anthracnose-like disease was observed on fruit of T. kirilowii (cv. Wanlou9) in four fields (0.9 ha) located in Taihu county, Anhui Province of China. Approximately 60% of the T. kirilowii plants were affected in the fields. The symptoms initially consisted of small off-white necrotic spots, and later became larger, irregular gray necrotic lesions on green fruit, causing fruit rot and sometimes fruit drop. More than 10 symptomatic fruits were sampled, and small pieces of diseased tissue were surface sterilized in 0.1% HgCl2 for 2 min, 75% ethanol for 45 s, rinsed three times with sterile distilled water, then placed on potato dextrose agar (PDA) and incubated at 25℃in the dark. A fungus was consistently (80%) isolated from symptomatic fruit samples.Aerial mycelia were light gray, and radially black with white in reverse medium. Conidia were rarely produced on PDA, but prolific on water agar. The conidia were cylindrical to clavate, both ends rounded, had obvious circular granule in the center, and ranged from 14.6 to 19.9 μm × 5.4 to 7.3 μm. The morphological characteristics were similar to the descriptions of C. liaoningense by Diao et al. (2017). For molecular identification, representative isolates LG5-4 and LG9-6 were selected. Genomic DNA was extracted, and the internal transcribed spacer (ITS) region, actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-tubulin (TUB2) genes were amplified by PCR (White et al. 1990; Duan et al. 2018), and sequenced bidirectionally. A BLAST search of GenBank revealed the ACT and TUB sequences had 95.83% (KP890097), 99.20%, 95.33% (KP890111) and 99.84%, respectively, to C. liaoningense CAUOS2. A phylogenetic analysis was conducted using MEGA7 based on concatenated sequences of the four genes, indicating that the isolates were closely clustered with reference strains of C. liaoningense (98% bootstrap value). The two strains were deposited in the China General Microbiological Culture Collection Center as CGMCC3.20344 and CGMCC3.20345, and their sequences deposited in GeneBank (Accession nos. MW082811-12, MW117926-31), respectively. Pathogenicity tests were conducted on healthy fruit of T. kirilowii (cv. Wanlou9) using the wound inoculation by pinpricking and droplet (106 conidia/mL) on fruit surface. The experiments were done with three fruit per isolate (LG5-4 and LG9-6), and replicated three times. The controls were inoculated with sterile water. The fruit were covered with plastic bags and kept in a chamber (>90% RH, 28 to 30°C) for 14 days. Typical symptoms of yellow-brown lesions appeared 14 days after inoculation. No symptoms were observed on the controls. The fungus was re-isolated from the diseased tissues and identified as C. liaoningense by sequencing of the four genes, confirming Koch's postulates. C. liaoningense has been reported to cause anthracnose of mango and chili in China (Diao et al. 2017; Li et al. 2019). To our knowledge, this is the first report of C. liaoningense causing anthracnose on T. kirilowii. Due to cultivation of T. kirilowii in the region, further studies are required to develop management strategy of this disease.

First report of Pectobacterium versatile causing blackleg of potato in Serbia.

Potato blackleg is frequently observed on the production fields in the Bačka region of Vojvodina province, which is one of the largest potato-growing areas in Serbia. This disease usually occurs during June and July. In July 2020, blackleg symptoms in the form of stem necrotic lesions, vascular discoloration, hollow stems, and wilting of whole plants were noted on potato cultivar VR808 on a field 28 ha in size located in Maglić village (GPS coordinates 45.349325 N, 19.542768 E). Disease incidence was estimated at 20-25%. Isolations were performed from 12 potato samples on Crystal Violet Pectate medium (CVP). Stem sections consisted of brown lesions and healthy tissue (c.10 cm) were surface sterilized with ethyl alcohol 70% (w/v) and rinsed with sterile distilled water. Small pieces of tissue were taken at the edges of stem lesions (between healthy and diseased tissue) were soaked in phosphate buffer saline for 20 min and plated using a standard procedure (Klement et al. 1990). Single colonies that formed pits after 48 hours at 26 °C were re-streaked onto Nutrient Agar (NA) where creamy white colonies with smooth surfaces were formed. A total of 30 isolates were selected and DNA isolated from the colonies was further analyzed by polymerase chain reaction (PCR) using the partial dnaX gene (DNA polymerase subunit III gamma/tau) with primer pair dnaXf/dnaXr for Pectobacterium and Dickeya species identification (Slawiak et al. 2009). A single characteristic band of 535 bp was amplified in all isolates (Slawiak et al. 2009). DNA sequence alignment showed two distinct groups of isolates (Fig.S1), which were genetically uniform within each group. Using BLASTn search, it was established that the dnaX sequence of the first group (consisting of 19 Serbian potato isolates) had 99.79% identity with NCBI-deposited Pectobacterium versatile strains 14A and 3-2 from potato from Belarus (Acc. No. CP034276 and CP024842, respectively) as well as SCC1 from Finland (Acc. No. CP021894). The remaining 11 dnaX sequences had 100% identity with Pectobacterium carotovorum subsp. carotovorum strain CFBP7081 originating from water in Spain (Acc. No. MK516961). The partial dnaX sequences of three Serbian P. versatile isolates (Pv1320, Pv1520, and Pv1620) and one P. carotovorum subsp. carotovorum (Pcc2520) were deposited in GenBank under Acc. No. MW839571, MW805306, MW839572, and MW805307, respectively. These results, indicating combined infection in the observed field, signify the first identification of P. versatile in Serbia. Multilocus sequence analysis (MLSA) performed with proA (proAF1/ proAR1) and mdh (mdh2/mdh4) genes (Ma et al. 2007; Moleleki et al. 2013) grouped three tested Serbian potato P. versatile isolates together with P. versatile strains from NCBI (Fig.S2). For both tested genes, BLASTn search revealed 100% homology with P. versatile strain SCC1 from Finland. Three Serbian P. versatile potato isolates were deposited under Acc. Nos. MZ682623-25 for proA and MZ682620-22 for mdh genes. According to the routine tests suggested for Pectobacteriaceae (Schaad et al. 2001), Serbian isolates possessed microbiological traits identical to P. versatile description (Portier et al. 2019). Pathogenicity was performed on potato cultivar VR808 with three selected P. versatile isolates (Pv1320, Pv1520, and Pv1620) in the following assays: (i) surface-sterilized tuber slices with holes in the center filled with 100 µL of bacterial suspensions (adjusted to 109 CFU mL-1) to test the isolates' ability to cause soft rot, and (ii) young, four-week old plants with developed 3rd true leaf (c. 30 cm tall) were inoculated by injecting stems with bacterial suspension adjusted to 107 - 108 CFU mL-1 at a height 5 cm above the soil line. Negative controls were treated with sterile distilled water. Inoculated plants were kept under controlled conditions (25 °C temperature and >70% relative humidity). Each assay was replicated twice. Soft rot appeared on tuber slices 24 h after inoculation. On inoculated stems, initial symptoms manifested as greasy elongated spots at inoculation sites two days after inoculation (DAI), and subsequently extended along the vascular tissue and became necrotic. Whole plant's decay was recorded in five DAI, while negative controls remained healthy. To complete Koch's postulates, bacteria were re-isolated from symptomatic potato plants and confirmed by PCR and sequencing of dnaX. This first report of P. versatile in potato indicates that blackleg currently present in Serbia is caused by a diverse bacterial population. This pathogen was first identified in genome comparison as 'Candidatus Pectobacterium maceratum' (Shirshikov et al. 2018) and was later renamed as Pectobacterium versatile sp. nov. (Portier et al. 2019). Thus far, bacterium Pectobacterium carotovorum subsp. brasiliensis has been recognized as dominant pathogen on most of the infected fields in Vojvodina province, and was recently noted on one plot subjected to a combined infection with Dickeya dianthicola (Marković et al. 2021). Findings achieved in this study are highly relevant, as they point to the diversity in potato blackleg pathogens, likely due to the increasingly widespread distribution of imported seed potatoes.

First Report of Fusarium sacchari Causing Sugarcane Wilt in Bangladesh.

Wilt is one of the destructive fungal diseases of sugarcane (Saccharum officinarum L.) affecting sugarcane production in many sugarcane growing regions including Bangladesh. In July 2018, sugarcane plants showing wilting symptom including withered, dried leaves and shriveling stalks with brown to pinkish discoloration in internal tissues (Viswanathan, 2013) were collected from sugarcane field from Gazipur district (23°54'41″ N, 90°23'20″ E) of Bangladesh. Average disease incidence was 17% in 60 ha of sugarcane fields that were sampled for diseased plants with visible symptoms. Ten infected stalks (30cm) were collected from five severely infected fields. The stalks were surface sterilized with 70% ethanol for 1 minute followed by 0.2% sodium hypochlorite for 3 minutes and then rinsed three times with distilled water. Small pieces (2mm×2mm) of the inner discolored tissue were cut with a strile scalpel and placed on half strength Potato Dextrose Agar (PDA) medium followed by incubation for 7 days at 25 °C. A total of ten isolates were recovered from the infected sugarcane stalks and were purified by single spore culture method. Isolates produced floccose and cottony colony with white mycelia and dark violet pigmentation on the lower surface of the PDA plate. Microconidia were oval, generally, without septation measuring 5-9.6 µm length and 1.8-3.7 µm width in size (Appendix 1). Macro conidia were not found on the PDA medium. Conidiophores produced mono- or poly-phialides and conidia were arranged in a false head on conidiophore which is consistent with Fusarium sacchari (Duan et al. 2019). For molecular identification, DNA was extracted from two representative isolates BTFSS1 and BTFSS6. The internal transcribed spacer (ITS) region, translation elongation factor 1-α (TEF-1α) gene and RNA polymerase II (RPB2) genes were amplified using primers ITS1/ITS4, EF1α-F EF1α-R and 5F2/11aR, respectively (Appendix 2). Obtained ITS sequences (GenBank accession nos. MT176492.1 and MT177209.1) showed 99.8% similarity with the sequences from F. sacchari strain olowEKT1 (MK072727.1). Sequence identity of TEF-1α (GenBank accession nos. MW558264.1 and MW507839.1) was 99.36% with the sequence of F. sacchari strain FF031 (MK152501.1) and RPB2 (GenBank accession nos. MW558265.1 and MW507840.1) was 99.42% with the sequence of F. sacchari strain FF001 (MK152508.1), respectively. The isolates BTFSS1 and BTFSS6 were identified as F. sacchari based on the sequences alignment of ITS, TEF-1α and RPB2 and onolecular phylogenetic analyses by maximum likelihood tree method. To confirm the pathogen as a causal agent of wilt of sugarcane, pathogenicity tests were conducted in sugarcane plants by the plug method described by Viswanathan et al. (2011). Ten plants (variety BSRI Akh 42) were inoculated with 1 × 105 conidia mL-1 suspension of the isolate BTFSS1. A borehole was made on the third or fourth internode from the base of the sugarcane plant in where the conidial suspension was placed and the bore place was sealed with cane tissues. Control plants were inoculated with deionized distilled water. All canes were cuts longitudinally to evaluate the disease symptoms at fifteen and thirty days after inoculation. Inoculated stalks exhibited tissue discoloration that were similar to those observed in infected sugarcane field, whereas the control remained unaffected (Appendix 1). Pathogens were re-isolated from the artificially inoculated cane and characterized morphologically. Sugarcane is the second important cash crop and the only source of white sugar in Bangladesh (Rahman et al. 2016). Our report for the first time confirmed that F. sacchari is the causal pathogen of sugarcane wilt in Bangladesh. This report has significance to develop suitable management practices to control the pathogen in sugarcane field.

A Comparative Study of Biological Sample Preparation Methods Conventional Cryopreservation of Human Tissue

Background/Aims: Data that compare the effectiveness of methodologies for cryopreservation of human tissue are very limited. Thus, two different biological sample preparation methods, the conventional cryopreservation of human ovarian tissue using either spontaneous or initiated (so-called “seeded”) ice formation, was carefully investigated and compared. Materials and Methods: Biopsies of ovarian tissue were obtained from women with indications for chemotherapy or radiotherapy, and small pieces of experimental tissue (0.5 mm × 1 mm × 1–3 mm) were randomly distributed into three different groups: group 1 immediately after biopsy, experimental pieces after cryopreservation (thawing) with spontaneous ice formation (Group 2) and cryopreservation with initiated ice formation (Group 3). The pieces of tissue were cultured in vitro for 16 days, after which follicle viability and hormonal activity were evaluated. The level of statistical significance was set at P &lt; 0.05. Results: The obtained results indicated that culture supernatants for Groups 1, 2, and 3 showed estradiol 17-ß concentrations of 476, 465, and 459 pg/mL, respectively. Whereas pProgesterone concentrations were 9.68, 5.77, and 5.61 ng/mL, respectively. In addition, the mean primordial follicle density per mm3 for Group 1 was 12.1 ± 3.9, 3.1 ± 1.4 for Group 2 and 6.0 ± 2.3 for Group 3. Moreover, it was recognized that 91%, 16%, and 87% follicles for Groups 1, 2, and 3, respectively, were normal (P 2-1, 3 &lt; 0.05; P 1-3 &gt; 0.1). Conclusions: The obtained results revealed that for the best results using cryopreservation of human ovarian tissue, the protocol of conventional cryopreservation must include a step of initiated ice formation. Moreover, an advanced analytical detection technique of high sensitivity, mass spectrometry, can further be used in the future for an accurate determination of hormonal levels and other related compounds and for screening of possible biomarkers using the best-obtained sample preparation method.

Dose-Dependent Degeneration of Leydig Cells Following Kisspeptin-10 Administration: An Ultrastructural Study.

The discovery of kisspeptin signaling as a key regulator of gonadotropin- releasing hormone (GnRH) secretion from the hypothalamus enhanced our understanding of the neuroendocrine regulation of mammalian reproduction. Effects of central and peripheral administration of kisspeptin on plasma gonadotropins, testosterone, and spermatogenesis are studied in detail. The present study was conducted to check the ultrastructure of Leydig cells in prepubertal male rats in response to the administration of a range of kisspeptin doses. We administered a range of kisspeptin-10 doses (1 μg, 1 ηg, and 10 ρg) intraperitoneally to prepubertal male Sprague-Dawley rats (PND 35) twice daily after every 12 hours. Control rats were injected with physiological saline in parallel. At the end of the treatment, plasma concentrations of testosterone were measured by competitive binding radioimmunoassay, and small pieces of rat testicular tissue were processed for electron microscopy to examine the ultrastructure of Leydig cells. Plasma testosterone concentration was reduced significantly at 1ηg (P<0.05) and 1μg (P<0.01) doses as compared to control. Distinct ultrastructural changes categorized as dilatation of cytoplasmic organelles, irregularly shaped nuclei with nuclear membrane invaginations, reduced nuclear sizes, degeneration, and vacuolation were observed in the kisspeptin-10 treated Leydig cells as compared to control. Quantification of the data showed reduced Leydig cell indices and hyperplasia of the interstitial cells. It is concluded that chronic intermittent administration of kisspeptin-10 has a dose-dependent degenerative effect on the plasma testosterone levels and Leydig cells ultrastructure in prepubertal male rats.

First Report of Dickeya fangzhongdai causing soft rot in Orchids in Canada.

Dickeya fangzhongdai was originally described as the causal agent of bleeding canker of pear tree in China. Recently, D. fangzhongdai was isolated and identified as the causal agent of soft rot in an orchid plant purchased in a local supermarket in Prince Edward Island, Canada. A water-soaked dark green spot on the leaf surface was observed and later became larger soft rot symptom. The origin of the orchid plants was traced back to a producer in Ontario, Canada who propagated them from with cuttings originally imported from the Netherlands and Taiwan. Bacterial isolations were made from a soft rot lesion on an orchid leaf by surface sterilization of small pieces of marginal tissue of the diseased leaf in 70% alcohol. The small pieces of leaf tissue were then washed three time using sterile water, and immersed in drops of sterile water. Bacterial streaming was observed under the microscope and non-fluorescing bacterial colonies were isolated on King's B and casamino acid-peptone-glucose agar plates and purified as isolates 908, 909, 910 and 911. The DNA samples were extracted from the four isolates, as well as the diseased leaf tissue, and tested by using a qPCR assay with the specific primer/probe set (DfF/DfR/DfP) for D. fangzhongdai (Tian et al. 2020). The assay yielded PCR amplicons of 135 bp with a melting temperature of 86.5±0.6 °C as did two control reactions using genomic DNA from D. fangzhongdai strains JS5T and QZH3 originally isolated in China, providing presumptive identification of the orchid isolates as D. fangzhongdai. To fulfill Koch's postulates, freshly purchased healthy orchid plants (n=4) were inoculated by leaf injection with the bacterial isolates obtained in this study and strains JS5 T and QZH3 at ~107 CFU/ml. Three leaves of the same side of the plants were inoculated with the same strains as triplicates. Sterile water was used as the negative control. Inoculated plants were incubated in a growth chamber with a 16 h photoperiod at 23 °C. Water soaked lesions developed in 3-5 days after inoculation followed by soft rotting in leaves inoculated with the new bacterial strains from orchid plants while strain QZH3 caused soft rot in 10 days after inoculation (Fig. S1). The non-fluorescing bacteria on King's B plates with colony morphology similar to those inoculated were re-isolated from the inoculated leaves and confirmed to be D. fangzhongdai by qPCR. Phylogenetic analysis of the assembled 16S rRNA sequence of isolate 908 (GenBank accession number: MT984340), together with GenBank data of all Dickeya spp. and some Pectobacterium spp, using neighbor-joining (NJ) method inferred with MEGA X software (Kumar et al. 2018) showed that isolate 908 clustered with strains JS5T and QZH3 at a phylogenetic distance of 0.0007. This clearly indicated that isolate 908 and JS5T and QZH3 belong to the same genus. Species-level identification of isolate 908 was achieved by genome sequencing and analysis based on average nucleotide identity (ANI). Genomic DNA of isolate 908 was sequenced with Illumina MiSeq to provide approximately 180X genome coverage. After quality checking using FastQC (Andrews 2010), de-novo assembly was performed with VelvetOptimiser v2.2.6 (Zerbino and Birney 2008). The draft genome size of strain 908 was 4,938,027 bp consisting of 76 contigs with 56.8% G+C content and 63,801 bp as N50. The draft genome was checked for misassembled fragments using QUAST v5.0.2 (Gurevich et al. 2013) and found to be of good quality. The draft genome sequence is deposited in GenBank under the accession number of JADCNJ000000000. The draft genome sequence of strain 908 was compared to that of D. fangzhongdai JS5T type strain genome using FastANI v1.2 (Jain et al. 2018) resulting in an ANI value of 98.9%, which is above the 95% cut-off for the same species. Previously, it was reported that D. fangzhongdai caused soft rot in orchid in Europe (Alič et al. 2018) and in onions in New York (Ma et al. 2020). The difference in virulence among D. fangzhongdai strains warrants further investigation and their pathogenicity on potato is being investigated to evaluate any threat to the potato industry. To our knowledge, this is the first report of D. fangzhongdai causing soft rot disease on orchids in Canada and North America.

First Report of Colletotrichum cliviae Causing Anthracnose of Rubber Tree in China.

Rubber tree (Hevea brasiliensis) is the most important species producing commercially viable natural rubber latex, which is an important raw material for many industrial uses. Anthracnose caused by Colletotrichum is one of the most severe diseases of rubber tree, especially in China. However, the diversity of Colletotrichum from rubber trees is complex and needs further identification (Liu et al. 2018). In January 2021, anthracnose-like lesions were observed on the leaves of rubber seedlings at a plantation in Qiongzhong City (19.08 N, 109.54E), Hainan Province, China. The lesions initially produced dark brown spots on the leaves of rubber trees, and then gradually evolved into larger necrotic spots. The diseased leaves shrank and died until they fell off. Small pieces of symptomatic leaf tissues were soaked in 0.5% mercuric chloride for 40 s, washed with sterile water for three times, and plated on potato dextrose ager (PDA). The plates were incubated at 28°C with 12-h light/dark regime for 10 days. Two isolate HN16 and HN18 from different leaves were subcultured on PDA using the single-spore method. The center of the colony is brown or grayish brown with the leading edge of growth in culture being white. Conidia were hyaline, septate, and cylindrical, with obtuse ends (14-20 μm × 4-6 μm).These morphological features confirmed the identity of the two isolated fungi as Colletotrichum cliviae (Yang et al. 2009). To confirm the morphological identification, five loci informative for identification of Colletotrichum spp. were amplified and sequenced, including the 5.8S nuclear ribosomal gene with the two flanking internal transcribed spacers (ITS), beta-tubulin (TUB), actin (ACT), an intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and chitin synthase (CHS-1), using the primer pairs ITS-1F/ITS4,T1/Bt-2b, ACT-512F/ACT-783R, GDF/GDR and CHS-79F/CHS-345R, respectively (Weir et al 2012). Sequences were deposited in NCBIs GenBank database (MW971911-MW971920). A multilocus phylogenetic analysis revealed that the sequences of the two isolates and published sequences of C. cliviae S37 clustered into one clade with a 100% confidence level. The two isolates were determined as C. cliviae by combining morphological and molecular characteristics. To verify pathogenicity, six leaves of rubber plants at the light green phase were sprayed with drop of spore suspension (106 conidia/ml) and sterile distilled water as control. After inoculation, all leaves were maintained in a climate box at 28°C with 100% relative humidity. After 3 days, lesions similar to those observed with dark brown spots in the field appeared on the inoculated leaves, while the controls remained symptomless. The fungus was reisolated from inoculated leaves and confirmed as C. cliviae by morphological and molecular identification, fulfilling Koch's postulates. The pathogen has been mainly reported on soybean in Brazil and China. To our knowledge, this is the first report of anthracnose on rubber tree caused by C. cliviae in China. However, Colletorrichum gloeosporioides and Colletorrichum acutatum are the two major causatives agent of anthracnose of rubber trees in China (Cai et al. 2016; Shi et al. 2019). The disease caused by C. cliviae was observed only at one rubber plantation with its distribution needing further investigation.

First Report of Sea buckthorn Stem Wilt Caused by Fusarium sporotrichioides in Gansu, China.

Sea buckthorn (Hippophae rhamnoides) is an important deciduous shrub for fruit and ecological restoration in arid and semi-arid regions of China. Twelve Chinese and Russian cultivars (cv. Shenqiuhong, eshi01, ... eshi11) were planted about 1.6 acre area in a seedling nursery, located in Qingyang City of Gansu province in northwest China, where high mortality (more than 70%) of sea buckthorn was observed in late July 2019. Symptoms consisted of massive chlorosis, drooping leaves and dried-up stems on 5-year-old trees. Pieces of tree roots and stems with irregular light-brown discoloration in the xylem vessels were selected. Small pieces of discolored tissue were surface disinfested (1 min in 1% sodium hypochlorite, followed by three rinses with sterile distilled water), air-dried, and placed on potato dextrose agar (PDA) medium for 5 days at 25°C in the dark. A fungus was consistently isolated from both diseased roots and stems tissues. Colonies on PDA grew rapidly. Dense mycelia were pinky-white initially, and became carmine red color with age on the undersurface of the plate. Macroconidia were moderately curved, 3 to 5 marked septa, hyaline, thick walled, and measuring 27.8± 3.6 µm × 4.8 ± 0.5 µm (n = 30). Microconidia were abundant, pear-shaped, ellipsoid to fusoid, often with a papilla at the base, and 8.4 ± 2.2 µm ×3.1 ± 0.3 µm (n = 30). Genomic DNA was extracted for amplification and sequencing of the internal transcribed spacer region (ITS1 and ITS4 primers) (White et al. 1990) of the ribosomal DNA (Accession Nos. MN160235 to MN160238) and translation elongation factor-1 alpha (EF1 and EF2 primers, accession Nos. MN429075 to MN429078) (O'Donnell et al. 1998). The sequences revealed 99% similarity to the sequences of the ITS (AY188917), and 100% identity with EF1-α (JF740808) regions of Fusarium sporotrichioides. Based on morphological and molecular characteristics, the fungus was identified as F. sporotrichioides (Leslie and Summerell 2006). Koch's postulates were fulfilled on healthy, potted 1-year-old sea buckthorn seedings using two isolates in a greenhouse at 25 °C, 90% relative humidity, and 12-hour light/dark photoperiod. Ten potted seedings were inoculated on the stems by placing a 5-mm-diameter mycelial plug (5-day-old PDA cultures for each isolate) into the surface of a wound created with a needle, and the inoculation sites were covered with Parafilm to maintain moisture. Ten seedings were inoculated with PDA plugs as controls. Seven to ten days after inoculation, typical symptoms of dark-brown necrotic lesions on chlorotic leaf margins were observed. About 2 weeks after inoculation, the inoculated stems were gradually dry up, accompanied by withering and fallen leaves. Control plants remained asymptomatic. Pathogens were successfully isolated from the inoculated stems again, exhibiting morphological characteristics identical to those of F. sporotrichioides. Previous papers reported F. sporotrichioides as a common pathogen caused lavender wilt (Cosic et al. 2012), foliar spots on forage corn (Moya-Elizondo et al. 2013) and maize ear rot (Wang et al. 2019). To our knowledge, this is the first report of sea buckthorn stem wilt caused by F. sporotrichioides on several Chinese and Russian cultivars in Gansu province of China. In Heilongjiang province, the same disease was reported in 2010 (Song et al. 2010), nearly 30 longitudes away from Gansu province. Therefore, this disease appears to be a serious risk for future sea buckthorn production.

First report of Phaeoacremonium amygdalinum associated with almond dieback and wood disease in Italy.

In the last 10 years, almond (Prunus dulcis (Mill.) Webb) cultivation areas in the region of Apulia (southern Italy) have increased. A recent survey in young almond orchards recorded characteristic disease symptoms including rapid collapse of branches, chlorosis of leaves (with sudden wilting and death) in June/July, and bud and shoot dieback in December-March. Internal wood symptoms on stems consisted of brown vascular streaking that led to death of young apical shoots within a few weeks. Wood samples were collected and taken to the laboratory, where they underwent disinfection according to Fisher et al. (2002). Fungal isolation techniques were applied, with small pieces of wood tissue (0.5-1.0 cm) placed on potato dextrose agar (PDA; Oxoid Ltd.) supplemented with 300 mg/L streptomycin sulphate, and incubated at 23 ±2 °C in the dark. After 10 days, based on morphological features carried out by microscopic studies, 85% (n = 55) of the resulting fungal colonies (N = 65) were preliminarily attributed to known fungal pathogens of woody tissues, of which 12.4% (n = 8) belonged to the Phaeoacremonium genus. These last were purified by transferring single germinating conidia onto fresh PDA plates. In particular, the conidiophores were mostly short, usually unbranched, erect to flexuous, up to 5-septate, brown to pale brown, verruculose on the lower part, (12-)15.7-41(-55) (av. -29) μm long and 1.6-3.2 (av. 2.1) μm wide (n = 30). Conidia hyaline, oblong ellipsoidal-obovoid or subcylindrical (3-)4-5 μm long and 1.5-3 (av. = 4.4 × 2.1) μm wide (n =30). All Phaeoacremonium isolates were successfully subjected to genomic DNA extraction, according to Carlucci et al. (2013). Partial actin and β-tubulin genes were amplified with the ACT-513F/ACT-783R (Carbone and Kohn 1999) and T1 (O'Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995) primers, respectively. Consensus sequences were compared with reference strains (extype) in the GenBank database, using the Basic Local Alignment Search Tool (BLAST). In particular, all isolates showed 99-100% similarity with reference strains of Phaeoacremonium amygdalinum Gramaje, Armengol & L. Mostert (JN191301; JN191305). Actin and β-tubulin sequences of P. amygdalinum strain Pm10 were deposited in GenBank (MW716265; MW714562). In spring 2020, artificial inoculations with two isolates of P. amygdalinum (Pm10; Pm15) were carried out on 10 healthy, 2-year-old almond seedlings of cv. 'Filippo Cea'. Agar plugs (diameter, 0.3-0.5 cm) taken from 10-day-old cultures grown on PDA at 23 ±2 °C were inserted into small wounds under the bark of the stems (length, 0.4-1.0 cm) made with a sterile scalpel. After inoculation, the wounds were wrapped with wet sterile cotton wool and sealed with Parafilm. Ten almond seedling were used as controls, being inoculated with sterile agar plugs. The experiment was replicated three times. The inoculated young almond plants were grown in pots in a greenhouse without temperature control. After 150 days, the inoculated plants showed decline symptoms and internal longitudinal brown wood streaking (average, length, 2.7-6.4 cm). Phaeoacremonium amygdalinum was reisolated from symptomatic wood of 95% of the experimental almond seedlings, thus fulfilling Koch's postulates. No symptoms were observed on almond seedling used as controls. Phaeoacremonium amygdalinum was first described as a fungal pathogen of almond in Spain (Gramaje et al., 2012). To the best of our knowledge, this is the first report of P. amygdalinum associated with almond dieback disease in Italy. References Carbone I. and Kohn L.M. 1999. Mycologia 91:553. Carlucci A. et al. 2013. Phytopathol. Mediterr. 52:517. Fisher et al 2002. Mycol. Prog. 1:315 Glass N. L. and Donaldson G. C. 1995. J. Cl. Microbiol. 41:1332. Gramaje et al. 2012. Persoonia 28:1. O'Donnell K. and Cigelnik E. 1997. Mol. Phylogenetics Evol 7:103.

Correlation between insulin-like growth factor 1 gene expression and fennel (Foeniculum vulgare) seed powder consumption in muscle of sheep

It has been shown that addition of fennel in the diets of domestic animals has positive and beneficial effects on growth and meat production traits. Thus, the purpose of current study was to investigate the effect of adding fennel in the ration on growth characteristics and on insulin-like growth factor 1 (IGF1) gene expression in muscle tissue of Kermani lamb. Feeding of animals were performed with three levels of fennel including zero, 10 and 20 g/kg dry matter (DM) for 90 days. After slaughter, small pieces of tissues were removed and rapidly transferred to a nitrogen tank. Then, total RNA extracting and the Real-Time PCR reaction was performed. Results showed that as the level of fennel in the diet increases the amount of IGF1 gene expression also increases significantly in humeral muscle and femur (leg) muscle tissues (p < 0.05). In animals fed with fennel, femur muscle weight, back muscle weight, lean meat weight, final weight, warm carcass weight and live daily gain were greater than in animals fed with diet without fennel (p < 0.05). According to the findings of this investigation, it can be concluded that fennel, by creating positive effects on IGF1 gene expression can be used to improve muscle structure.

Establishment of Patient-Derived Organoid Culture Platform of Mantle Cell Lymphoma

BackgroundMantle Cell Lymphoma (MCL) is a rare incurable non-Hodgkin's lymphoma despite remarkable recent therapeutic innovations such as CAR T cell therapies. Drug sensitivity and immunotherapy efficacy assays using preclinical tumor models are important in new therapy development. As for preclinical tumor models, in addition to the commonly used mouse model such as patient-derived xenograft (PDX) and genetically engineered mouse models, a 3 rd tumor model, the patient-derived organoid (PDO) in 3D culture model has been established in many types of solid tumors. However, no PDO models have been generated from MCL yet. We have established MCL PDO model by optimizing cell isolation, culture add-ons, spatial and temporal conditions. Our protocol presents a versatile MCL PDO platform, suitable for quick drug screens and applicable for rapid immunotherapy evaluation. For proof of concept demonstration, we tested the therapeutic efficacy of CD19-targeted CAR T-cell in MCL-specific PDOs. The established PDO procedures can be used for diverse biopsies including whole blood, apheresis, bone marrow, lymph node and previously established PDX tumors.MethodsFor human blood, bone marrow and apheresis biospecimens, we first eliminated red blood cells using RBC lysis buffer. The cells were then spun down and cell pellets were resuspended in culture medium and aliquoted and spun-down into V-shaped 96-well plates at 1-6´10 6 cells per well. After overnight incubation, the cell culture medium was replaced by 60 ml of precooled 60% Matrigel per well. The 3D formed aggregates were gently transferred into 24-well plate and feed the solidified domes with the medium containing an in-house cytokine cocktail. The developed organoids reach the drug screening optimal phase within 3-6 days or it can be further expanded for future use. For PDX tumor and human lymph node biospecimens, we first sliced tumor tissue using surgical scalpel blade, which resulted in small tissue pieces that were then resuspended in HBSS. Debris (>100 mm) were removed by brief gravity sedimentation. The resultant tissue pieces were then resuspended in 60% Matrigel in culture medium and dispensed onto pre-warmed 24-well plates. The primary tumor organoids were observed for 3-5 days before drug testing on processed for further expansion. For organoid passaging, primary organoids were extracted in pre-cooled medium by mechanically breaking the gel domes. The extracted organoids were digested by collagenase/dispase. The resultant MCL and stromal cells were enforced to aggregate in V-shape wells as described in the apheresis procedure. Organoid cell composition was examined using FACS. Drug sensitivity and T-cell activity against the MCL organoids were assessed using CellTiter-Glo 3D kit.ResultsWe have successfully established and optimized the PDO procedure from diverse MCL biopsies (Fig. 1A). The success rate of MCL organoid from apheresis was > 80% with even higher success rates when using PDX tumors, lymph node and bone marrow samples (> 90%). One critical step for processing the biopsies is preserving the original stromal cells. FACS analysis showed that although <5% of total cell population is composed of macrophages, circulating fibroblast, epithelial and endothelial cells and they are indispensable for organoid formation and expansion. Addition of hematopoietic cytokines in culture medium significantly improved the organoid formation and expansion from diverse biopsies. FACS with lineage markers revealed that the cell populations of the primary and secondary organoids were not significantly different (Fig. 1B). Tumor-killing activity of the CD19-targeting CAR T cells was assessed by co-culturing the CAR T cells with MCL organoids.ConclusionWe have established an MCL PDO platform which is time-efficient, labor-saving, cost-effective and highly reproducible. This platform provides a rapid approach for immune cell activity assays and drug screening. The organoids have been successfully used to generate PDX models. This platform can also be used for investigating the mechanism of drug resistance in the context of different TMEs. [Display omitted] DisclosuresWang: Acerta Pharma: Consultancy, Honoraria, Research Funding; BioInvent: Research Funding; Pharmacyclics: Consultancy, Research Funding; Lilly: Research Funding; CStone: Consultancy; Oncternal: Consultancy, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; Genentech: Consultancy; OMI: Honoraria; Newbridge Pharmaceuticals: Honoraria; Hebei Cancer Prevention Federation: Honoraria; Moffit Cancer Center: Honoraria; Molecular Templates: Research Funding; Physicians Education Resources (PER): Honoraria; Mumbai Hematology Group: Honoraria; InnoCare: Consultancy, Research Funding; Anticancer Association: Honoraria; VelosBio: Consultancy, Research Funding; Loxo Oncology: Consultancy, Research Funding; DTRM Biopharma (Cayman) Limited: Consultancy; Juno: Consultancy, Research Funding; Epizyme: Consultancy, Honoraria; Bayer Healthcare: Consultancy; CAHON: Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; Celgene: Research Funding; Imedex: Honoraria; Kite Pharma: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Scripps: Honoraria; Dava Oncology: Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Clinical Care Options: Honoraria; Chinese Medical Association: Honoraria; BGICS: Honoraria; Miltenyi Biomedicine GmbH: Consultancy, Honoraria.

First report of Corynespora cassicola causing black spot on Sarcandra glabra in China.

Sarcandra glabra is an important Chinese medicinal plant, which was widely cultivated under forest in south China. Guangxi province is the main producing areas of this herb. In June 2019, a serious leaf disease was found causing severe defoliation in the S. glabra plantation under bamboo forest in Rongan country, Guangxi province (109°13'N''E). About 70% of the plants in the plantation (300 ha) showed the similar symptoms. Initially, circular lesions appeared on young leaves as black spots (about 1 to 2 mm). Then, the spots gradually enlarged usually with an obvious yellowish margin (6 to 8 mm). Finally, the lesions coalesced and formed irregular, black, and large necrotic areas, resulting in the leaf abscission. For pathogen isolation, small pieces of tissue (5×5 mm) taken from 25 diseased leaves were sterilized with 75% ethanol for 30 s, subsequently, soaked in 0.1% HgCl2 for 2 min, rinsed three times in sterile distilled water, dried, and then placed aseptically onto the potato dextrose agar (PDA) plates, and incubated at 28 °C (12 h/12 h light/dark). Three days later, the isolates were placed on a new PDA plate for subsequent purification and sporulation. 20 pure fungal isolates were obtained from single spores. Of which, 15 isolates showed similar morphological characteristics.The colonies on PDA were round, dense, gray edge and dark gray in center area. Conidia in culture were appeared light brown, cylindrical in shape, with 0 to 8 septa, and 55 to 165 μm × 5.2 to 13.5 μm in size (mean = 106.2 μm × 8.6 μm, n = 30). These morphological characteristics resemble those of Corynespora sp. (Berk. & M.A. Curtis) C.T. Wei (Ellis et al. 1971). A single-spore isolate (ZD5) was selected from the 15 fungal isolates for a subsequent molecular identification. The genes of internal transcribed spacer (ITS) of ribosomal DNA, β-tublin, and actin were amplified with the primer pairs ITS-1/ITS-4 (White et al. 1990), β-tubulin 2-Bt2a/Bt2b (Glass and Donaldson 1995), ACT-512F/ACT-783R (Carbone and Kohn 1999), respectively. And the ITS, β-tublin, and actin sequences were deposited in the GenBank database with the accession numbers MW362446, MW367029, and MW533122. Blast analysis and neighbor-joining analysis based on ITS, β-tublin, and actin sequences using MEGA 6 revealed that the isolate was placed in the same clade as C. cassicola with 100% bootstrap support. Pathogenicity test was performed on the two-year-old potted S. glabra. Six-mm-diameter mycelial plugs were attached to the healthy leaves of S. glabra for co-culture, while the control group was attached with PDA. All plants were covered with plastic bags for 2 days in order to maintain high humidity and cultured in a greenhouse at 28 °C with a 12-h/12-h light/dark cycle. The symptoms appeared 2 days after co-culture were identical to those observed in the field. The same fungus was re-isolated from the lesions, and further morphological characterization and molecular assays, as described above.The control leaves remained symptomless during the pathogenicity tests. According to the previous literatures, C. cassicola is a plant pathogenic fungus with a broad host range, which can damage diverse tropical plants including Salvia miltiorrhiza (Lu et al. 2019), Solanum americanum (Wagner and Louise 2019), Vitex rotundifolia (Yeh and Kirschner 2017), Cucumis sativus, Lycopersicon esculentum (Hsu et al. 2002), Carica papaya (Tsai et al. 2015),and so on. To our knowledge, this is the first report of C. cassicola causing leaf spot on S. glabra in China.

First report that Colletotrichum aenigma causes leaf spots on Aquilaria sinensis in China.

Aquilaria sinensis (Lour.) Spreng, also known as eaglewood, belongs to the Thymelaeaceae family and has a considerably high medicinal value. It has been enlisted as the class II national key protective plant. In June 2019, about 15 percent of A. sinensis treelets in a forest area of China's Hainan province were observed to have the anthracnose symptoms. The diseased spots on leaves of A. sinensis treelets were usually round or irregular with pale yellow edges. The color of the center of the lesion was firstly light brown and then black or yellowish-brown. Small pieces of tissue from the edge of the leaf spots were surface sterilized in 75% alcohol for the 60s, washed twice with sterile distilled water, and then cultivated at 28 °C in darkness on potato dextrose agar (PDA) medium. One fungus was systematically isolated to get pure cultures. The culturing of the three isolates was carried out in PDA media at 28 °C for a week. The average diameter of the collateral colony was 6.80 ±0.60 cm. Initially, the fungal colonies were white aerial mycelium and the central area of the colonies slowly turned jacinth. After seven days, the central mycelium turns grayish-green and the colonies' undersurfaces were grey to white. The colony's surfaces were fluffy and round with smooth edges. Conidia were cylindrical, smooth, and transparent, with a slight indentation in the middle and uneven distribution of small particles inside, 12.5-20.6×3.5-6.8 µm (ave=15.9±1.40×5.18±1.07, n=50). Appressoria were typically elliptic or irregular and brown to dark brown. The isolates were characterized as Colletotrichum gloeosporioides species complex on the basis of the conidial morphology and culture representation, (Deng et al. 2017; Weir et al. 2012). To further verify the identification of the species, CX-0301, the isolated representative strains were extracted for genomic DNA. mating type 1-2-1 (Mat-1-2-1) ApMat, actin (ACT) gene, chitin synthase (CHS), and beta-tubulin (TUB2) gene were amplified using the primer pairs VcaMat-5F/VcaMat-5R, ACT-512F/ACT-783R, CHS-1-79F/CHS-1-354R, and TUB2-T1/Bt2b, respectively (Damm et al. 2012; Du et al. 2005). The homologous sequences of MN310694, MN310693, MN310692, and MN310691 were submitted to GenBank. These genes have ≥a 97% sequence similarity to the genes of Colletotrichum aenigma (MG717319.1, MG717317.1, MH476565.1, MH853679.1, respectively) in GenBank. These morphological and molecular characteristics identified that the pathogen is C. aenigma. (Weir et al. 2012). To further verify the isolated pathogen, the pathogenicity test was performed on uninfected healthy 2-year-old eaglewood seedlings. The conidial suspension (1×106 conidia/ml) of 5ml was sprayed on both surfaces of 10 leaves of plants of the same age and height and the controls were treated solely with distilled water (Deng et al. 2017). Upon completion of inoculation, plants were kept under greenhouse conditions with an assigned temperature of 28 ± 2°C while keeping relative humidity to 90% on a 12-h fluorescent light/dark regime. Anthracnose-like symptoms were observed 6 days postinoculation. The control plant tissues remained healthy. Follow up reisolation of C. enigma culture was obtained in PDA agar plates from leaf infected lesions, and the morphological features were found to be consistent with that of CX-0301 isolate, satisfying Koch's postulates. Based on the characterized information, it is the first report of Colletotrichum aenigma responsible for causing leaf spots on Aquilaria sinensis in China. Thereby, this provides a theoretical reference for the research and control of anthracnose on A. sinensis.

Occurrence of Dickeya fangzhongdai Causing Soft Rot of Banxia (Pinellia ternata) in China.

Banxia [Pinellia ternata (Thunb.) Breit., Araceae] is a perennial herbaceous plant, where the tuber is commonly used in traditional Chinese herbal medicine. In the summer of 2020, an outbreak of soft rot of Banxia was observed in Zhugentan Town (30°50'N, 112°91'E), Qianjiang City, Hubei Province, with about 56% percentage of infected plants. Symptomatic plants initially appeared with small water-soaked spots on leaves that progressed into extensive translucent spots when facing a light source. The bacteria further spread to the stems and tubers. Infected tubers appeared normal, but inner macerated inclusions exuded when touched. The whole plant was macerated and collapsed within a few days. Ten leaves with typical symptoms were obtained from a diseased field, by surface sterilizing in 75% ethanol for 30 s and 0.3% NaClO for 5 min, washing the tissue sections three times in sterile water. Small pieces of tissue (5 × 5 mm) were removed from lesion borders, plated on nutrient ager medium, and cultivated at 37 ℃ for 48 h. Five representative isolates were selected for further identification. Colonies were all smooth and transparent. In addition, these strains were Gram-negative, and had the ability to reduce D-arabinose, melibiose, galactose, raffinose, rhamnose, inositol, and mannitol, but not reduce 5-keto-D-gluconate, L-xylose, amygdalin, and sorbitol. Genomic DNA was extracted from isolate stain ZG5. The 16S rDNA gene, recombinase A (recA) gene, and DNA polymerase III subunits gamma and tau (dnaX) were amplified by PCR with the primers 27f/1492r (Weisburg et al. 1991), recF/recR (Waleron et al. 2002), and dnaXf/dnaXr (Sławiak et al. 2009), respectively. The PCR products were sequenced, then submitted to GenBank (GenBank MW332472, MW349833, MW349834, respectively). BLAST search showed that the sequences of 16S rDNA, recA, and dnaX respectively matched ≥99% with D. fangzhongdai strains DSM 101947 (CP025003), QZH3 (CP031507), and PA1 (CP020872). Pathogenicity tests were performed on 10 healthy, 3-month-old P. ternate plants. Five plants were injected with 20 μl of bacterial suspension (108 CFU/ml) of isolate ZG5, and other plants were injected with sterile water as a negative control. All tested plants were incubated at 28 ℃ and individually covered with a plastic bag. After 24 h, soft rot symptoms all appeared on the pathogen-inoculated leaves, whereas no symptoms on the control leaves. The pathogenicity test was repeated three times and obtained same results. Koch's postulates were fulfilled by reisolating D. fangzhongdai from inoculated plants. Meanwhile, PCR were performed on the reisolated bacteria as above described, and the pathogen was identified and confirmed as D. fangzhongdai. Here we report that D. fangzhongdai causes soft rot of P. ternata in China. The disease progressed very rapidly, and reduced the yield and quality of tubers. Thus, more research is needed to implement effective strategies to manage this disease.

First Report of Strawberry Crown Rot Caused by Xanthomonas fragariae in China.

Strawberry (Fragaria × ananassa Duch.) is a kind of fruit with great economic importance and widely cultivated in the world. From 2019 to 2020, a serious crown rot disease was sporadically observed in several strawberry cultivars including 'Zhang Ji', 'Hong Yan' and 'Yue Xiu' in Shanghai, China. Initially, water-soaked rot appeared in inner tissue of strawberry crown, then progressed into browning and hollowing symptoms accompanied with yellow discolorations of young leaves. To isolate and identify the causal agent, small pieces of tissue taken from ten diseased crowns were sterilized by 70% alcohol. The cut-up pieces were macerated and serially diluted. The dilutions were placed on nutrient agar (NA) medium. After incubation at 25°C for 4-5 days, the yellow bacterial colonies were tiny and were streaked on NA plate for purification. The colonies were yellow, mucoid, smooth-margined, and five independent representative colonies were used for further confirmation. To confirm the species identity of the bacterial, genomic DNA was extracted from the five representative isolates, and 16S rRNA gene was amplified and sequenced using universal primers 27F/1492R. The 16S rRNA sequence was deposited in GenBank (MW725235) and showed 99% nucleotide similarity with Xanthomonas fragariae strain LMG 708 (NR_026318). The isolate's identity was further confirmed by X. fragariae-specific primers XF9/XF12 (Roberts et al. 1996). All five isolates could be detected by XF9/XF12 primer. To confirm Koch's postulates, five healthy strawberry plants were placed in 1000 ml glass beakers by submerging the cutting wound in 50 ml the bacterial suspension of 108 CFU/ml. Five additional strawberry plants treated with sterilized water served as a control. The beakers containing inoculated plants were sealed with plastic film at 25°C. Water-soaked rot appeared on internal tissue of crown similar to those observed in the field within 10-12 days after inoculation, while the control samples remained healthy. The bacteria were re-isolated from rot of inoculated crowns, and confirmed by X. fragariae-specific primers XF9/XF12. X. fragariae has been reported to cause angular leaf spot on strawberry in China (Wang et al. 2017; Wu et al., 2020). It's also found that X. fragariae could systematically infect crown tissue (Milholland et al. 1996; Mahuku and Goodwin, 1997). To our knowledge, this is the first report of X. fragariae causing strawberry crown rot in China. This report increased our understanding of X. fragariae, and showed that the spread of this disease might seriously threaten the development of strawberry industry in the future.

First Report of Colletotrichum boninense Causing Anthracnose on Rosa chinensis in China.

Chinese rose (Rosa chinensis Jacq.) is cultivated for edible flowers in southwestern China (Zhang et al. 2014). In March 2020, a leaf spot disease was observed on about 3-5% leaves of Chinese rose cultivar 'Mohong' in Guizhou Botanical Garden (26°37' 45'' N, 106°43' 10'' E), Guiyang, Guizhou province, China. The symptomatic plants displayed circular, dark brown lesions with black conidiomata in grey centers on leaves, and leaf samples were collected. After surface sterilization (0.5 min in 75% ethanol and 2 min in 3% NaOCl, washed 3 times with sterilized distilled water) (Fang 2007), small pieces of symptomatic leaf tissue (0.3 × 0.3 cm) were plated on potato dextrose agar (PDA) and incubated at 28oC for about 7 days. Two single-spore isolates, GZUMH01 and GZUMH02, were obtained, which were identical in morphology and molecular analysis.Therefore, the representative isolate GZUMH01 was used for further study.The pathogenicity of GZUMH01 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 (106 spores ml-1) onto leaves until runoff, and the control leaves sprayed with sterile water. The plants were maintained at 25°C with high relative humidity (90 to 95%) in a growth chamber. The pathogenicity test was carried out three times using the method described in Fang (2007). The symptoms developed on all inoculated leaves but not on the control leaves. The lesions were first visible 48 h after inoculation, and typical lesions similar to those observed on field plants after 7 days. The same fungus was re-isolated from the infected leaves but not from the non-inoculated leaves, fulfilling Koch's postulates. Fungal colonies on PDA were villiform and greyish. The conidia were abundant, oval-ellipsoid, aseptate, 15.8 (13.7 to 18.8) × 5.7 (4.3 to 6.8) µm. The fungal colonies, hyphae, and conidia were consistent with the descriptions of Colletotrichum boninense Moriwaki, Toy. Sato & Tsukib. (Damm et al. 2012; Moriwaki et al. 2003). The pathogen was confirmed to be C. boninense by amplification and sequencing of the internal transcribed spacer region (ITS), the glyceraldehyde-3-phosphate dehydrogenase (GADPH), actin (ACT), and chitin synthase 1 (CHS-1) genes using primers ITS1/ITS4, GDF1/GDR1, ACT512F/ACT783R, and CHS-79F/CHS-345R, respectively (Damm et al. 2012; Moriwaki et al. 2003). The sequences of the PCR products were deposited in GenBank with accession numbers MT845879 (ITS), MT861006 (GADPH), MT861007 (ACT), and MT861008 (CHS-1). BLAST searches of the obtained sequences of the ITS, GADPH, ACT, and CHS-1 genes revealed 100% (554/554 nucleotides), 100% (245/245 nucleotides), 97.43% (265/272 nucleotides), and 99.64% (279/280 nucleotides) homology with those of C. boninense in GenBank (JQ005160, JQ005247, JQ005508, and JQ005334, respectively). Phylogenetic analysis (MEGA 6.0) using the maximum likelihood method placed the isolate GZUMH01 in a well-supported cluster with C. boninense. The pathogen was thus identified as C. boninense based on its morphological and molecular characteristics. To our knowledge, this is the first report of the anthracnose disease on R. chinensis caused by C. boninense in the world.

First Report of Moldy Core of Sweet Tango Apples from New Zealand Caused by Alternaria arborescens.

First Report of Moldy Core of Sweet Tango Apples from New Zealand Caused by Alternaria arborescens.