Lesion Margins Research Articles

Role of Multiparametric Magnetic Resonance Imaging of the Brain in Differentiating Neurocysticercosis From Tuberculoma.

The two most common infectious causes of ring-enhancing lesions are neurocysticercosis (NCC) and tuberculoma. It is a challenge to differentiate NCC and tuberculomas radiologically since they show the same imaging findings on computed tomography (CT). Hence, this study was performed to assess the role of magnetic resonance imaging (MRI) as an additional advanced modality to aptly characterize the lesion. Conventional MRI with additional advanced imaging sequences like diffusion-weighted imaging (DWI), apparent diffusion coefficient (ADC), magnetic resonance spectroscopy (MRS), and post-contrast T1-weighted imaging (T1WI) aids in characterizing the lesion and helps in differentiating NCC and tuberculomas. To compare the findings of DWI, ADC cut-off values, spectroscopy, and contrast-enhanced MRI in differentiating NCC from tuberculoma. Individuals who matched the inclusion criterion underwent an MRIof the brain (plain and contrast) in a 1.5 Tesla, 18-channel, magnetic resonance scanner (Magnetom Avanto®, Siemens Healthineers, Erlangen, Germany). The following imaging sequences were included: T1WI (axial and sagittal), T2-weighted imaging (axial and coronal), fluid-attenuated inversion recovery, DWI at 0, 500, and 1000 mm2/sb-values with corresponding ADC values, and single-voxel MRS. Based on MRI features such as number, size, location, margins of lesions, scolex, surrounding edema, DWI features with corresponding ADC values, enhancement pattern of lesions, and spectroscopy findings, we evaluated and differentiated the lesions as NCC or tuberculoma. Radiological diagnoses were correlated in terms of clinical symptoms and response to treatment. In our study, 42 subjects were included, of which the total number of NCC cases was 25 (59.52%) and tuberculoma was 17 (40.47%). The mean age of patients included was 42.85 ± 14.76 years (21 to 78 years). On post-contrast imaging, all 25 cases of NCC (100%) showed thin ring enhancement whereas the majority of tuberculomas (64.7%) showed thick irregular ring enhancement. On MRS, all 25 cases (100%) of NCC showed an amino acid peak and all 17 cases (100%) of tuberculoma showed a lipid lactate peak. On DWI, out of 25 NCC cases, restriction of diffusion was absent in the majority of cases (88%) and out of 17 cases of tuberculoma, restriction of diffusion was present in 12 cases (70.5%) (T2 hyperintense tuberculoma, indicative of caseating tuberculoma with central liquefaction) and was absent in the rest. In our study, the mean ADC value of NCC lesions (1.30 ± 0.137 x 10-3 mm2/s) was found to be greater than that of tuberculoma (0.74 ± 0.090 x 10-3 mm2/s). ADC value of 1.2 x 10-3 was obtained as a cut-off to differentiate NCC and tuberculoma. The ADC cut-off value of 1.2 x 10-3 mm2/s showed a sensitivity of 92% and specificity of 94.1% in differentiating NCC from tuberculoma. Conventional MRI with additional advanced imaging sequences likeDWI, ADC, MRS, and post-contrast T1WI aids in characterizing the lesion and thereby helps in differentiating NCC and tuberculomas. Hence, multiparametric MRI assessment is useful in making a prompt diagnosis and eliminating the need for a biopsy.

First report of Botryosphaeria dothidea causing leaf spot of Camellia oleifera in China.

Camellia oleifera Abel., a small evergreen tree or shrub, is mainly distributed in central and southern China with a larger scale of 4.5 × 106 hectares (Zhu 2020). In May 2021, severe leaf spots were observed in plantation located in Shuangfeng County (27°41'36" N, 111°56'60" E), Hunan Province, China. More than 60 C. oleifera plants were surveyed with over 80% disease incidence. The symptoms on leaves were initially small brown lesions from leaf margins or tips, developing to suborbicular or irregular-shaped dark brown lesions, leading to leaves withered. A total of 60 symptomatic samples were randomly collected. Lesion margins were surface sterilized in 2% sodium hypochlorite for 1 min, rinsed with sterile distilled water for three times, dried, placed on potato dextrose agar (PDA), and incubated at 25°C in the dark for 3 days. Hyphal sections from colony edges were transferred to new PDA plates. Three isolates of Botryosphaeria dothidea were obtained. Colonies of B. dothidea were initially white gradually turning dark-gray with dense aerial mycelium after 6 days. To induce sporulation, colonies of YCB17 were transferred to synthetic nutrient-poor agar (SNA) with sterilized leaves of C. oleifera. Cultures were initially incubated at 25°C in the dark for 3 days, then alternatively exposed to 12-hours near-UV light and 12-hours white light (CHU et al. 2021). After 5 days, conidia formed on leaves were examined microscopically. The conidia were unicellular, aseptate, hyaline, and fusoid, 20.9-25.5×4.7-6.4 µm (n = 50). Morphological characteristics of the isolates matched the description of B. dothidea (Slippers et al. 2014). DNA sequence was amplified using primer pairs ITS1/ITS4 (Tang et al. 2022), EF1-728F/986R (Slippers et al. 2004), and βt2a/2b (Glass & Donaldson. 1995) respectively. The sequences of three isolates (YCB2, YCB3, YCB17) were deposited in GenBank with accession numbers ON714603, MZ613350, MZ613349 (ITS), OM328342, OM328343, OM328344 (TEF-1α), and OM328345, OM328346, OM328347 (TUB2). A blast search of sequences showed the ITS, TEF-1α, and TUB2 sequences had >99% identity with homologue sequences from B. dothidea isolates IRNHM-KZ49 (MG198191.1), CAP288 (EF638732.1) and Mu1 (MK423987.1), respectively. For pathogenicity testing, healthy leaves of 2-year-old C. oleifera plants in the greenhouse were spray-inoculated with conidial suspension (2×106 conidia/mL) from YCB17. Ten surface-sterilized and wounded leaves per plant were sprayed with 30 µL suspension. The other ten wounded leaves sprayed with sterile distilled water served as control. All plants were kept in the greenhouse with temperature at 26 ± 2°C and 50% relative humidity. After 12 days, initial symptoms were observed on more than 80% leaves inoculated with conidial suspension, whereas no symptoms were observed on the control leaves. The test was repeated three times with similar results. It was found that B. dothidea could cause leaf spot of C. oleifera. The infected leaves showed same symptom as samples. Re-isolated fungi from infected leaves were morphologically identical to B. dothidea. Botryosphaeria dothidea has been reported causing leaf spot in a wide range of hosts, but has not previously been reported causing disease on C. oleifera. To our knowledge, this is the first report of B. dothidea causing leaf spot of Camellia oleifera in China. The information on identification of this fungus may be helpful to the control and prevention of the disease.

First Report of Diaporthe eres Causing Leaf Spot on Pseudocydonia sinensis in China.

Pseudocydonia sinensis is a Chinese ornamental plant with great landscaping value. Its fruit is additionally used for medicinal purposes (Lim 2012). In June 2020, a leaf spot disease was observed in the campus of Nanjing Forestry University (32°04'34.53″N 118°48'42.06″E). The symptoms began with irregular red-brown spots, which gradually enlarged, extended and overlapped, with an incidence of 85% (29/34 trees). Pieces of leaf tissue (3 to 4 mm²) from the lesion margins were surface-sterilized with 75% ethanol for 30 s and 1% NaClO for 90 s. Subsequently, the tissues were rinsed with sterile H2O, placed on potato dextrose agar (PDA) medium and incubated at 25℃ for 5 days. The same fungus was isolated from 90% of tissues. Pure cultures were obtained by monosporic isolation.The representative isolate NJMG 5-7 was used for morphological and molecular characterization. The growing fungal colony on PDA was initially white, but gradually turned grey. Pycnidia formation was observed on PDA supplemented with alfalfa stems. The pycnidia produced two different types of conidia, α and β, which ooze out in yellow creamy mucilaginous masses. Conidiophores were hyaline, cylindrical and smooth, 16.8 to 33.1 × 1.5 to 2.6 μm (n=30). Conidiogenous cells were 13.6 to 29.3 × 1.5 to 2.7 μm (n=30). The α-conidia were, unicellular, hyaline elliptical or fusiform, bi-guttulate, 6.5 to 9.2 × 1.8 to 3.3 µm (n = 50). The β-conidia were hyaline, aseptate, without guttules, filiform, curved, with obtuse ends, 12.5 to 25 × 1.0 to 1.8 µm (n = 50). To verify species identity, the partial sequences of the internal transcribed spacer (ITS) region, and calmodulin (CAL), translation elongation factor 1 alpha (EF1-a), and beta-tubulin genes (TUB) were amplified from isolate NJMG 5-7 with primers ITS1/ITS4 (White et al. 1990), CAL-228F/CAL-737R (Carbone & Kohn 1999), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The sequences were deposited in GenBank (OP223050 for ITS, OP252809 for CAL, OP252807 for EF1-a, and OP252808 for TUB). A BLAST search of GenBank showed that ITS, CAL, EF1-a and TUB sequences of NJMG 5-7 were similar to those of D. eres CBS 138594 (99% identity), AR5193 (99%), AR5193 (99%) and MG281193 (100%), respectively. The morphological and molecular results identified the isolate as D. eres (Feng et al. 2015). To fulfill Koch's postulates, a pathogenicity test was conducted using three P. sinensis plants. Six leaves from each tree were wounded and inoculated with mycelial plugs (about 4 mm in diameter) of D. eres from a 3-day-old culture grown on PDA. Inoculations with sterile PDA plugs on different leaves of the same tree were used as controls. All inoculated leaves were enclosed in plastic bags together with a wet cotton ball inside. Sterile H2O was sprayed into the plastic bags to keep moisture conditions. Five days later, all inoculated points showed lesions similar to those previously observed in the field, whereas controls were asymptomatic. The pathogen was successfully reisolated from the inoculated symptomatic parts on PDA and identified from its morphology, thus fulfilling Koch's postulates. This fungus can cause a variety of diseases. To our knowledge, this is the first report of D. eres causing leaf spots on P. sinensis in the world. These findings provide a foundation for future studies on the epidemiology and control of this newly emerging disease.

A Case of Subcutaneous Schwannoma of the Scalp

Schwannomas are benign neural tumors derived from Schwann cells, which surround the peripheral nerves. Here, we present a case of subcutaneous schwannoma of the scalp, with the goal of increasing awareness of the differential diagnosis of scalp masses. A 45-year-old woman was admitted with a palpable scalp mass on the occiput initially noticed 2 years prior. Palpation revealed a 1.5-cm round subcutaneous mass, which was soft and exhibited a movable tendency. The lesion margin was well-circumscribed and contained under the skin. Computed tomography showed a 6 × 12-mm soft-tissue density subcutaneous nodule in the right paramedian occipital region. Thus, an epidermal cyst or lipoma was suspected. The pathological diagnosis (hematoxylin and eosin staining) revealed evidence indicating a subcutaneous schwannoma of the scalp. We report a subcutaneous schwannoma of the scalp, demonstrating that schwannomas can also occur as subcutaneous scalp lesions. Therefore, we suggest that although the treatment of subcutaneous schwannomas does not differ from other soft-tissue masses, a pathological examination should be performed to establish an exact diagnosis in such cases.

Accuracy of Artificial Intelligence-Based Automated Quantitative Coronary Angiography Compared to Intravascular Ultrasound: Retrospective Cohort Study.

An accurate quantitative analysis of coronary artery stenotic lesions is essential to make optimal clinical decisions. Recent advances in computer vision and machine learning technology have enabled the automated analysis of coronary angiography. The aim of this paper is to validate the performance of artificial intelligence-based quantitative coronary angiography (AI-QCA) in comparison with that of intravascular ultrasound (IVUS). This retrospective study included patients who underwent IVUS-guided coronary intervention at a single tertiary center in Korea. Proximal and distal reference areas, minimal luminal area, percent plaque burden, and lesion length were measured by AI-QCA and human experts using IVUS. First, fully automated QCA analysis was compared with IVUS analysis. Next, we adjusted the proximal and distal margins of AI-QCA to avoid geographic mismatch. Scatter plots, Pearson correlation coefficients, and Bland-Altman were used to analyze the data. A total of 54 significant lesions were analyzed in 47 patients. The proximal and distal reference areas, as well as the minimal luminal area, showed moderate to strong correlation between the 2 modalities (correlation coefficients of 0.57, 0.80, and 0.52, respectively; P<.001). The correlation was weaker for percent area stenosis and lesion length, although statistically significant (correlation coefficients of 0.29 and 0.33, respectively). AI-QCA tended to measure reference vessel areas smaller and lesion lengths shorter than IVUS did. Systemic proportional bias was not observed in Bland-Altman plots. The biggest cause of bias originated from the geographic mismatch of AI-QCA with IVUS. Discrepancies in the proximal or distal lesion margins were observed between the 2 modalities, which were more frequent at the distal margins. After the adjustment of proximal or distal margins, there was a stronger correlation of proximal and distal reference areas between AI-QCA and IVUS (correlation coefficients of 0.70 and 0.83, respectively). AI-QCA showed a moderate to strong correlation compared with IVUS in analyzing coronary lesions with significant stenosis. The main discrepancy was in the perception of the distal margins by AI-QCA, and the correction of margins improved the correlation coefficients. We believe that this novel tool could provide confidence to treating physicians and help in making optimal clinical decisions.

Surgical margin mapping for lentigo maligna and lentigo maligna melanoma: traditional technique (visual inspection with dermoscopy) versus combined paper tape and reflectance confocal microscopy technique.

The detection of subclinical margin in lentigo maligna/lentigo maligna melanoma (LM/LMM) can be challenging for the dermatologist. Reflectance confocal microscopy (RCM) enables to observe in vivo atypical melanocytes beyond the clinical margins. The aim of this study is to establish which of these methods (clinical examination and dermoscopy versus "Paper tape - RCM") is more precise to define the lesion margin and to reduce the number of re-intervention and overtreatments in cosmetically sensitive areas. Fifty-seven cases of LM/LMM were analyzed during 2016-2022. Pre-surgical mapping procedures in 32 lesions were effectuated with dermatoscopy. Furthermore, pre-surgical mapping procedures in 25 lesions were effectuated with RCM and paper tape. RCM method's accuracy to detect subclinical margins was 92.0%. In 24 of 25 cases, the lesions were excised completely during the first intervention. In 20 of 32 cases analyzed with dermoscopy, a second surgical intervention was effectuated. The RCM paper method allows us to delineate subclinical margin more accurately and reduce overtreatment, especially in sensitive areas, such as the face and neck.

First report of leaf spot disease caused by Xenodidymella glycyrrhizicola on Glycyrrhiza glabra

Glycyrrhiza glabra (liquorice) is a perennial shrub belonging to the Fabaceae family. It is a native of Mediterranean areas and primarily cultivated for its extracts which are used in the pharmaceutical and food industries, and in the manufacture of food supplements (Hayashi & Sudo, 2009). Several foliar diseases, such as powdery mildew, rust and leaf spots, have been reported to endanger the long-term viability and production of liquorice (Farr & Rossman, 2023). In 2022, a leaf spot disease was observed on c. 40% of the two-year-old plants in a liquorice field in Ankara, Turkey. Leaf spots were brown-to-black in colour, sometimes with a light brown centre. Lesions coalesced to large areas of blighted tissue with age (Figure 1a). Branches and petioles also became covered in oval lesions (Figure 1b). Pieces (5 × 5 mm) from lesion margins were surface sterilised in 70% ethanol (30 seconds) and 1% NaOCl (1 minute) and rinsed three times with sterile water. Phoma-like colonies were isolated from surface-sterilised parts of diseased tissues on potato dextrose agar (PDA). One representative isolate (Xg_Gg01), purified using the hyphal tip method, was selected for molecular, morphological and pathogenic characterisation and transferred to plates containing fresh PDA, oatmeal agar (OA), and malt extract agar (MEA) for seven days at 25°C with 12 hr light and 12 hr dark cycles. Colonies on MEA reached about 45 mm in diameter, margin regular and white, brown towards the centre, which was brown and orange due to conidial discharge from pycnidia (Figure 2a). Colonies reaching about 50 mm in diameter on the OA were marginally regular and olivaceous, with a colour change to black due to pycnidia formed towards the centre (Figure 2b). Colonies on the PDA reached about 43 mm in diameter, and the margin was regular and pale olive; the middle and centre were black due to dense pycnidia, and light brown conidial discharge was observed in the centre (Figure 2c). Pycnidia were mostly superficial or immersed in agar, ostiolate, 230–470 × 190–365 μm, and globose or subglobose and brown, becoming irregular-shaped and black with age (Figure 3a). Conidia were aseptate, variable in size and shape, oblong, broadly ellipsoidal, hyaline, becoming pale brown with age and measured 6.1 ± 9.3 × 3.4 ± 6.4 μm (Figure 3b). The internal transcribed spacer (ITS) of ribosomal DNA, partial 𝛽-tubulin gene (TUB2) and RNA polymerase second largest subunit (RPB2) loci of isolate Xg_Gg01 were amplified and bidirectionally sequenced with the universal primer sets; ITS1/ITS4 (White et al., 1990), BT2a/ BT2b (Glass & Donaldson, 1995), and 5f2 (Reeb et al., 2004)/7cr (Liu et al., 1999), respectively. A BLAST search of the resultant sequences (GenBank Accession Nos. OQ568230 for ITS, OQ571627 for TUB2, and OQ571628 for RPB2) showed 99.61–100% nucleotide identity with the holotype strain CBS 684.97 of X. glycyrrhizicola from New Zealand. The combined maximum-likelihood tree of ITS, TUB2, and RPB2 regions with 1000 bootstrap replicates showed that the isolate from the current study was clustered with strains of X. glycyrrhizicola derived from GenBank with strong support (Figure 4). Koch's postulates were fulfilled by spraying 10 ml of a spore suspension (106 spores/ml prepared in a 0.1% Tween-20 solution) of isolate Xg_Gg01 on each of five two-month G. glycyrrhizicola plants in trial pots. The plants were covered with plastic bags for 24 hr post inoculation. After seven days, spots were observed on the inoculated plants’ leaves and petioles (Figure 1c). No symptoms were observed on control plants, sprayed with sterile distilled water only. A morphologically identical X. glycyrrhizicola was isolated from affected tissues. To the best of our knowledge, this is the first report of X. glycyrrhizicola associated with leaf spots on G. glabra (Farr & Rossman, 2023). The fungus was previously reported on Glycyrrhiza lepidota in Iran (Ahmadpour et al., 2022) and New Zealand (Hou et al., 2020). However, G. glabra constitutes a new host record for this pathogen in Turkey and worldwide.

Leaf Spots of Salix babylonica Caused by Colletotrichum gloeosporioides s.s. and C. siamense Newly Reported in China.

Salix babylonica L. shows a great potential for restoration of contaminated water or soils and has a high ornamental value (Li et al. 2015). In mid-October 2021, a leaf spot disease, with an incidence of approximately 61%, occurred on leaves of 25-year-old S. babylonica on the campus of Nanjing Forestry University. On average, 65% of the leaves per tree were infected. Symptoms began as dark brown, irregular spots, and the centers were grayish white. The spots gradually enlarged with time. Fresh specimens were collected from 3 trees (10 leaves/tree). Small tissue pieces cut from lesion margins were surface-sterilized (Mao et al. 2021), plated on potato dextrose agar (PDA), and incubated at 25°C. Three representative isolates (NL1-7, NL1-10, and NL1-13) were obtained and deposited in The China Forestry Culture Collection Center. The colonies of 3 isolates were white, grayish white at the center. The conidia of 3 isolates were one-celled, straight, subcylindrical, hyaline, smooth, 14.6-18.6 × 4.3-6.7 µm, 13.8-16.7 × 4.7-6.0 µm and 12.1-16.9 × 5.4-7.5 µm (n = 50) for NL1-7, NL1-10, and NL1-13, respectively. The conidiophores of NL1-7 were hyaline to pale brown, septate, and branched, 18.9-48.0 µm (n = 50). Appressoria were one-celled, ellipsoidal, brown or dark brown, thick-walled. The conidiophores and appressoria of the other two isolates were almost identical to NL1-7. Based on morphological characteristics, the 3 isolates matched the Colletotrichum gloeosporioides species complex (Weir et al. 2012). DNA of the 3 isolates was extracted. The internal transcribed spacer region (ITS), actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin 2 (TUB2) loci were amplified using the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CL1C/CL2C, CHS-79F/CHS-354R, GDF1/GDR1, and T1/Bt2b, respectively (Weir et al. 2012). The sequences were deposited in GenBank [Accession Nos. ON870951 and ON858477 to ON858481 for NL1-7; ON908707 and ON858482 to ON858486 for NL1-10; ON870949 and ON858487 to ON858491 for NL1-13]. BLAST result showed that ITS, ACT, CAL, CHS-1, GAPDH, and TUB2 sequences of NL1-7 were identical to C. gloeosporioides at a high level (>99%). The sequences of NL1-10 and NL1-13 were consistent with C. siamense at a high level (>99%). A maximum likelihood and Bayesian Inference analyses using IQtree v. 1.6.8 and MrBayes v. 3.2.6 with the concatenated sequences (ITS, ACT, CAL, CHS-1, GAPDH, and TUB2) placed NL1-7 in the clade of C. gloeosporioides sensu stricto and NL1-10 and NL1-13 in the clade of C. siamense. To confirm their pathogenicity, 9 healthy 3-yr-old seedlings, and 10 leaves/seedling were wounded with a sterile needle and inoculated with 10 µL of conidial suspension (106 conidia/mL) of the 3 isolates, respectively. Three control plants were treated with sterile water. Seedlings were covered with plastic bags after inoculation and kept in a greenhouse at 25 ± 2°C and RH 80%. Within 7 days, all inoculated leaves showed lesions similar to those in the field, and controls were asymptomatic. C. gloeosporioides s.s. and C. siamense were reisolated from the infected tissues. It was reported that Colletotrichum species can cause many plant diseases, for example, C. acutatum causes twig canker (Swain et al. 2012), and C. salicis causes willow anthracnose (Okorski et al. 2018), etc. However, some Colletotrichum species are endophytic (Martin et al. 2021) and may only become pathogenic under the right conditions. This is the first report of C. gloeosporioides s.s. and C. siamense causing leaf spots on S. babylonica in the world. These data will help select appropriate strategies for managing this disease and further studies on the pathogen and the host.

First report of Fusarium sporotrichioides causing Fusarium fruit rot on peaches in Pakistan.

Peach (Prunus persica) is an important stone fruit crop cultivated on 14,350 hectares in temperate areas of Pakistan, where post-harvest fungal rots cause major economic losses for growers. During April-August 2019, rotted peaches were observed in fruit markets of Rawalpindi district (33° 37' 33.8052'' N and 73° 4' 17.1912'' E), Punjab Province, Pakistan. Symptomatic fruit were surface sterilized for 3 min in 0.5% NaClO and 1 min in 70% ethanol, then rinsed three times in sterilized distilled water and air-dried for 30 minutes on filter paper. Tissue segments were excised from the margins of rot lesions and placed on potato dextrose agar (PDA), followed by incubation for one week at 25 +1o C. Furthermore, to maintain original sporodochial morphology and conidial description tissue segments were also grown on carnation leaf agar (CLA) and synthetic nutrient agar (SNA) (Sever et al., 2012). A total of 22 isolates were obtained from different fruit markets; colonies were fast-growing with pinkish-white, fluffy mycelia. Microconidia were abundant, 7.5 to 11.3 × 2.1 to 4.1 μm, oval to spindle-shaped, thin-walled, and hyaline whereas, macroconidia were thick-walled, sickle-shaped, 5- to 7-septate, hyaline, and 23.5 to 39.7 × 3.7 to 7.0 μm on carnation leaf agar. Virulence intensity of fungal isolates was confirmed after pathogenicity. Based on morphological characteristics, isolates were identified provisionally as Fusarium sporotrichioides (Leslie and Summerell 2006). For molecular identification, three target gene regions (ITS, elongation factor (EF), and β-tubulin benA) were amplified for highly pathogenic fungal isolates (FUS21K, FUS9SM, and FUS63KP) (O'Donnell et al. 2015). BLAST searches of the obtained sequences indicated 100% homology with GenBank accession number MN452643, and 99.56% homology with MT635298 for the ITS region, similarly 100% identity with GenBank accession numbers MN555016, MN555126, and MN555037 for the EF gene, and 100% homology with GenBank accession numbers MK435577, MH791363, and GQ915448 for β-tubulin benA of the F. sporotrichioides species complex (O'Donnell et al. 2015). Sequences were deposited in GenBank; accession numbers for each gene were as follows: ITS, ON180670, ON180671, and ON180672; EF, ON312093, ON312094, and ON312095; and β-tubulin benA, ON312090, ON312091, and ON312092. Maximum likelihood analysis on RPB1 and RPB2 nucleotide sequences of various fusaria was conducted which revealed that F. sporotrichioides formed a monophyletic relationship to terminal fusarium clade (TFC) comprising 22 strongly supported species complexes and 10 monotypic lineages, which were provisionally recognized as Fusarium. To complete Koch's postulates, 10μl aliquots of spore suspensions (106 spores/ml) of each FUS21K, FUS9SM and FUS63KP were pipetted individually onto six ripe, asymptomatic peaches. Sterile distilled water was applied as a negative control on four fruit. Fruit were incubated at 25 ± 1°C for one week in a sterile moist chamber, and the trial was conducted three times. Rot symptoms associated with pinkish-white fluffy mycelia were observed on inoculated fruits after 72 hours, whereas no mycelium was observed on the negative controls. The cultures on PDA, CLA and SNA from each of the inoculated fruit were morphologically identical to the original culture. The pathogen was confirmed as a member of F. sporotrichioides species complex based on pinkish-white mycelium, with red color on the underside of petri plates and the manner in which conidia are born on conidiophores. This is the first report of F. sporotrichioides on peaches from Pakistan, providing an important foundation for peach growers, agricultural advisers, plant pathologists, and plant breeders to improve the management of this economically damaging disease.

V10-08 THE USE OF FOCAL IRREVERSIBLE ELECTROPORATION FOR PROSTATE CANCER

You have accessJournal of UrologyCME1 Apr 2023V10-08 THE USE OF FOCAL IRREVERSIBLE ELECTROPORATION FOR PROSTATE CANCER John Torosyan, Amy Cherry, Hasan Abbas, Shima Tayebi, and Abhinav Sidana John TorosyanJohn Torosyan More articles by this author , Amy CherryAmy Cherry More articles by this author , Hasan AbbasHasan Abbas More articles by this author , Shima TayebiShima Tayebi More articles by this author , and Abhinav SidanaAbhinav Sidana More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003328.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Focal therapy of prostate cancer is a treatment strategy that aims to destroy the cancer-containing parts of the prostate while preserving uninvolved prostatic tissue in an attempt to preserve genitourinary function. Irreversible Electroporation (IRE) is a novel ablative modality which utilizes electrical pulses to induce cell apoptosis and can be applied to various malignancies, including prostate cancer. The objective of this video is to discuss the mechanism of action of IRE and describe the components, setup, and operative technique for performing IRE for the focal treatment of prostate cancer. METHODS: IRE is performed under general anesthesia with the patient placed in lithotomy position. Monopolar electrodes are inserted into the prostate under ultrasound guidance and using a brachytherapy grid. The probes are placed 1.5 to 2cm away from one another, and 3-5mm away from the prostatic capsule using cognitive fusion to cover the lesion and 5-10mm margin. After initially estimating voltage configurations, ten test impulses are fired between each probe pair to assess resulting currents. Using test results, final voltages are assigned, and the remaining 80 impulses are fired between each probe pair. RESULTS: This case involves a 74-year-old male with a history of hypertension and hyperlipidemia who presented with an elevated Prostate-Specific Antigen (PSA) of 6.35 and was found to have Gleason 3+4 cancer in the Prostate Imaging Reporting & Data System (PI-RADS) 4 lesion localized in the right mid gland to apex. The patient had a pre-treatment International Prostate Symptom Score (IPSS) of 4-1 and a Sexual Health Inventory for Men (SHIM) of 18 without the use of erectile dysfunction medication. The patient underwent a successful IRE ablation of the lesion along with the margin. The procedure was tolerated well, and the patient was discharged later that day. At his 6-week follow-up, PSA declined to 0.66 and he had an unchanged IPSS/SHIM. CONCLUSIONS: The use of focal therapy in prostate cancer is promising because of its effectiveness paired with minimal procedural side effects. Focal IRE provides a safe and effective way to achieve focal therapy of prostate cancer. Source of Funding: None. © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e928 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information John Torosyan More articles by this author Amy Cherry More articles by this author Hasan Abbas More articles by this author Shima Tayebi More articles by this author Abhinav Sidana More articles by this author Expand All Advertisement PDF downloadLoading ...

Post-harvest Anthracnose of Carambola (Averrhoa carambola) Caused by Colletotrichum fructicola in China.

Carambola (star fruit), a popular fruit of Averrhoa carambola in many parts of the world, is considered to have many beneficial nutritional and medicinal effects (Lakmal, K., et al,2021). In March 2020, anthracnose disease was observed on carambola (about 15% of the fruit showed similar symptoms) in multiple local agricultural markets (113°36'E, 23°11'N) of the Yuancun district in Guangzhou, China. Initial symptoms of infected fruit samples appeared as water-soaked, brown lesions. As the disease progressed, numerous acervuli appeared on fruit surfaces. Salmon-colored spore masses were observed on some fruit. To isolate and identify the pathogen, small pieces (3-5 mm2) were excised from the lesion margins of the fruit, which were surface disinfested by 1% NaOCl (60 s), 70% ethanol (30 s) and then washed twice with sterile distilled water. After surface disinfestation, the tissues were cultured on potato dextrose agar (PDA). Pure cultures were obtained by transferring hyphal tips onto fresh PDA. Fungal isolates (YT-5/6/9) were obtained and the strain YT-5 was selected for further study. The colony of strain YT-5 grown on PDA for 7 days appeared to be cottony, white to pale gray with the presence of multiple masses of conidia. Conidia 13.5-20 × 4.8-6.5 μm (n = 50), hyaline, aseptate, straight and cylindrical with rounded ends. Perithecia were thick-walled and globose with a prominent, narrow neck. Asci 37.1-60.2 × 7.1-11.3 μm (n = 25), 4-8 spored, clavate to cymbiform. Ascospores 7.1-17.2 × 4.5-6.5 μm (n = 35), hyaline, large guttulate at the center, slightly curved with rounded ends. Based on the morphological characteristics, the strain was identified as Colletotrichum fructicola (Prihastuti et al. 2009). The molecular identity of the isolates was confirmed by sequencing the internal transcribed spacer (ITS) rDNA region, chitin synthase (CHS-1), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-tubulin (TUB2) genes (Prihastuti et al. 2009, Weir et al. 2012). BLASTN analysis of isolate YT-5 sequences, which were deposited in GenBank (ON428449, ON462353, ON886225, ON886224, ON462354) showed 100% identity with those of Colletotrichum fructicola (MW513778.1, MT918417.1, MW426526.1, MN525875.1, MT941526.1), respectively. A phylogenetic tree analysis based on the concatenated sequences confirmed the isolate YT-5 as C. fructicola. Pathogenicity tests were conducted on fresh fruit of carambola with the isolate YT-5. Healthy fruit was surface disinfested and inoculated with 5 mm mycelial discs of the strain YT-5 after being wounded with a needle or unwounded. Control fruit was inoculated with sterilized PDA plugs. All inoculated fruit was incubated at 26°C for 10 days post inoculation. Control fruit remained asymptomatic, whereas inoculated fruit developed symptomatic at the point of inoculation. The pathogenicity test was performed in duplicate. The pathogenic isolate of C. fructicola was successfully re-isolated on PDA from the symptomatic fruit, thus confirming Koch's postulates. C. fructicola has also been reported as a dominant and aggressive causal agent of anthracnose on sandy pear and avocado in China (Zhang et al. 2015; Li et al. 2022). To our knowledge, this is the first study to isolate and characterize C. fructicola causing carambola anthracnose and evaluate its pathogenicity in China, which will provide a better strategy for accurate diagnosis and effective management of anthracnose disease on carambola.

First Report of Leaf Spot and Blight on Crown Daisy Caused by Cercospora apii in China.

Crown daisy (Glebionis coronaria L.), also known as chrysanthemum greens, is a popular vegetable in Asia, especially in China. The leaves have been used in folk medicine as a tonic for the liver, blood, intestines and to control anemia and high blood pressure. In November 2020, severe leaf spot and blight was observed with 80% to 95% incidence on crown daisy growing in greenhouses in Fengxian, Shanghai, China(121°22'E, 30°53'N). Irregular rounded spots appeared with a light gray center and water-soaked margins. Round lesions enlarged and merged with age, followed by the development of a necrotic area resulting in the typical "frog-eye" and causing a continuous deterioration of crown daisy. Diseased leaves were washed in running water for 30 min. Small fragments (5 × 5 mm) taken from the margin of lesions were disinfected with 1% NaClO for 3 min, rinsed three times with sterile water, cultured on potato sucrose agar (PSA) augmented with 50 mg streptomycin/liter at 26 oC,and incubated in the dark. Colonies had identical morphology, and TH11290202 was selected and deposited in the plant pathology lab of Shanghai Academy of Agricultural Sciences. Mycelium was initially cottony and white and became appressed to the medium and dark brown with time. Conidia did not form on any media, including PSA, PDA, V8 agar (V8A), maize leaf carbonate agar (MLPCA), pepper leaf carbonate agar (PLPCA), etc. To confirm the identity of the pathogen, genomic DNA was extracted from TH11290202 with the cetyltrimethylammonium ammonium bromide (CTAB) method from the mycelia. Five loci were PCR amplified, namely, the internal transcribed spacer (ITS), translation elongation factor (TEF), calmodulin (cmdA), histone (H3) and actin (ACT), using primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Jaklitsch et al. 2005), CAL-F/CAL-R (O'Donnell et al. 2000), cylh3f/cylh3r (Glass and Donaldson 1995), and ACT-512F/ACT-783R (Carbone and Kohn 1999), respectively. The resulting sequences were deposited in GenBank (MW819910, MW981277, MW981278, ON798723, and MW981279). Analysis of the ITS, TEF, cmdA, H3 and ACT gene sequences of isolate TH11290202 revealed that it was a member of the genus Cercospora, sharing 99.79%, 99.66%, 98.10% 99.74% and 100% sequence similarity with type strain of Cercospora apii CBS 116455. A multilocus phylogenetic analysis was performed using sequences from other closely related taxa obtained from GenBank. Based on morphological and molecular characteristics, TH11290202 was identified as C. apii (Crous and Braun 2003; Groenewald et al. 2006; Milosavljević et al. 2014). To confirm pathogenicity, Koch's postulates were fulfilled on 30 mature plants, which were maintained in a growth chamber (at 26 °C, relative humidity 90%, 12/12 h light/dark). Surface-sterilized leaves were sprayed with a mycelial suspension. Brown lesions were formed 7 days after inoculation on 15 plants, whereas the noninoculated controls remained asymptomatic on the other 15 plants. To our knowledge, this is the first report of C. apii causing leaf spot and blight on G. coronaria in China and will provide useful information for developing effective control strategies.

First report of Diaporthe eres causing leaf spot disease on Machilus thunbergii in Korea.

Machilus thunbergii (Japanese bay tree) is native to warm temperate and subtropical regions in East Asia such as China, Japan, Korea, Taiwan, and Vietnam (Wu et al., 2006). This tree is used for landscape trees, windbreaks, and furniture because the wood is hard and dense (Hong et al., 2016). In May 2020, a leaf spot disease was observed on M. thunbergii in an arboretum on Wando Island, Korea. Among 25 trees surveyed in the arboretum, 7 trees showed 5 to 30% leaf spot disease. Symptoms consisted of gray and dry leaf spots up to approximately one to two centimeters in diameter, surrounded by a deep black margin. Leaf samples containing lesions were collected from the seven diseased trees. Pieces of leaf tissue (5mm × 5mm) were cut from the lesion margins and surface disinfected with 1% sodium hypochlorite (NaOCl) for 1 min and rinsed with sterile distilled water three times, patted dry on sterile paper towel and placed on Potato Dextrose Agar (PDA) in Petri dishes. From the cultures, ten fungal isolates were obtained and two representative isolates (CMML20-5 and CMML20-6) were stored at the Molecular Microbiology Laboratory, Chonnam National University, Gwangju, Korea. Colony morphology of the two isolates on PDA was observed after 7 days at 25°C in the dark. Conidiomata were induced after 7days in a 14h-10h light-dark condition using sufficiently grown mycelium in PDA, and both alpha and beta conidia were observed. Alpha conidia were 7.6 ± 0.9 × 2.8 ± 0.4 μm (n = 30), fusiform, aseptate, and hyaline. Beta conidia were 28.1 ± 3.6 × 2.7 ± 0.4 μm (n = 30), aseptate, hyaline, linear to hooked. Genomic DNA of the two isolates was extracted using the CTAB DNA extraction method (Cubero et al., 1999), followed by PCR using primer sets of the internal transcribed spacer (ITS1/ITS4) (White et al., 1990), elongation factor 1-α (EF1-728F/EF1-986R), calmodulin (CAL228F/CAL737R) (Carbone and Kohn, 1999), and TUB2 (Bt2a/Bt2b) (Glass and Donaldson 1995). PCR products were sequenced and analyzed to confirm species identity. The obtained sequences were deposited in GenBank (accession numbers OM049469, OM049470 for ITS, OM069429, OM069430 for EF1-α, OP130141, OP130142 for CAL, and OP130139, OP130140 for TUB2). BLASTn search analyses for ITS, EF1-α, CAL, and TUB2 sequences of two isolates selected resulted in near identical match (>97% for ITS, 100% for EF1-α, >99% for CAL, and >96% for TUB2) to sequences of Diaporthe eres strain AR4346 (=Phomopsis fukushii) (JQ807429 for ITS, JQ807355 for EF1-α, KJ435003 for CAL, and KJ420823 for TUB2). Phylogenetic analysis using maximum likelihood indicated that the two isolates grouped with reference strains (AR4346, AR4349, and AR4363) of D. eres with 76% bootstrap support. Based on morphological and phylogenetic analyses, the two isolates characterized in this study are members of the Diaporthe eres species complex as described by Udayanga et at. 2014. Pathogenicity tests were conducted using both detached leaf and whole plant assays. Mycelial PDA plugs (5-mm in diameter) or 10μl of 106 conidia suspensions were inoculated on detached leaves of M. thunbergii from 2-year-old trees and placed in 90 mm Petri-dishes containing wet filter papers or water agar medium. Mock inoculated controls used water in place of conidial suspensions. The plates were sealed with Parafilm and incubated at 25°C in the dark. Two year old M. thunbergii trees were inoculated with wet mycelia (1.5g) that was ground with a homogenizer and mixed with 50ml of sterile water and sprayed onto wounded leaves and stems with a needle. Mock inoculated controls were sprayed with water only. The inoculated seedlings were placed in plastic containers at 25 to 30°C to maintain high humidity. The pathogenicity tests were repeated three times with three replications. In detached leaves, symptoms of black spots were observed 6 days after mycelial plug inoculation and 20 days after conidia inoculation. In whole plants, typical symptoms were observed 9 days after inoculation. Symptoms were not observed on the control leaves and plants. Diaporthe eres was re-isolated from the inoculated leaf and whole plants and morphologically identified, fulfilling Koch's postulates. Diaporthe eres has been reported to cause a leaf spot on Photinia × fraseri 'Red Robin' in China (Song et al. 2019). To our knowledge, this is the first report of leaf spot disease caused by Diaporthe eres on Japanese bay tree (Machilus thunbergii) in Korea. It is expected that use of this tree will expand given its utility, however infection with D. eres can cause serious diseases to the leaves and stems. Therefore, further studies on disease management are needed.

First Report of Post-Harvest Anthracnose of Kiwifruit Caused by Colleotrichum fioriniae in Liaoning and Sichuan Province, China.

Kiwifruit (Actinidia spp.) is one of the most important fruit crops in China. Post-harvest anthracnose symptoms were observed on kiwifruit in October 2021. Kiwifruits 'Longcheng 2' (n=200) were obtained from an orchard in Kuandian city of Liaoning province, China (124°32'E, 40°20'N). And cultivar 'Cuiyu' (n=100) were harvested from orchards in Mianzhu city, Sichuan Province, China (104°03'E, 31°15'N). After storage at 24 °C and 80% relative humidity (RH) for 8 days, the disease incidence of 'Longcheng 2' and 'Cuiyu' was 30% and 15%, respectively. Symptoms of diseased fruits appeared as water-soaked, irregular and light brown lesions. Orangish conidial masses were observed on some fruits. Ten lesion margins (5×5 mm) from 'Longcheng2' or 'Cuiyu' were respectively excised, surface sterilized by 70% ethanol (1 min), 1% NaOCl (5 min), washed, dried, plated on potato dextrose agar (PDA), and incubated at 25 °C for 5 days. Eight isolates were obtained from 'Longcheng 2' (LC1-3 to LC1-10) and nine strains from 'Cuiyu' (CY1-2 to CY1-10). The representative isolates LC1-3 and CY1-2 were put on PDA, and appeared white to pale gray on the upper side. However, isolate LC1-3 secreted red pigments after 7 days of culture. Conidia of LC1-3 were hyaline, smooth-walled, single-celled, cylindrical (3.0 to 4.9×7.2 to 14.7 µm, n=50). Ellipsoidal single cell conidia of CY1-2 were hyaline, and ranged in size from 3.2 to 5.0×8.5 to 13.9 µm (n=50) born on conidiophores. Appressorium of isolates LC1-3 and CY1-2 were globose to ellipsoid with 4.2 to 7.4×7.3 to 10.8 µm and 3.0 to 4.9×6.3 to 10.3 µm in size, respectively (n=50) (Fu et al. 2019 ). Four genes (ACT, CHS, GAPDH, TUB2) and the ITS region were successfully amplified and sequenced from all isolates (Weir et al. 2012). Based on sequence alignment, the isolates from 'Longcheng 2' or 'Cuiyu' were identical. BLAST analysis of the ACT, CHS, GAPDH, ITS and TUB2 sequences of LC1-3 (ON018724, ON018722, ON018720, OM980324, ON018718) or CY1-2 (ON018725, ON018723, ON018721, OM980325, ON018719) showed high similarity with C. fioriniae (CBS 128517; JQ949613, JQ948953, JQ948622, MH865005, JQ949943) were 97.1% to 99.7% or 98.1% to 99.7%, respectively. Phylogenetic analysis using concatenated sequences (maximum likelihood method) with MEGA 11 showed LC1-3 and CY1-2 were located within the same clade with C. fioriniae. Previous studies showed that C. fioriniae was classified into three subclades (Damm et al. 2012; Fu et al. 2019). However, LC1-3 and CY1-2 were located within a new subclade, namely the subclade IV. To test pathogenicity, healthy and mature kiwifruits 'Donghong', 'Cuiyu', 'Xuxiang', 'Hayward' and 'Jinyan' were surface sterilized. Each un-wounded fruit was dropped with 10 μl conidial suspension (105 conidia/ml) on the fruit surface. All fruits were placed into a plastic box and stored at 24 °C under 80% RH. Each treatment consisted of 10 fruits and were repeated three times. After 8 days, typical anthracnose lesions were observed on all inoculated fruits. Whereas, the controls treated with sterile distilled water remained asymptomatic. The pathogens re-isolated from diseased fruits were similar morphological and identical to the original isolates, fulfilling Koch's postulates. Anthracnose caused by C. fioriniae has been reported on many fruits (Ling et al. 2020; Waller et al. 2021), but to our knowledge, this is the first report of anthracnose on kiwifruit caused by C. fioriniae. The results will provide valuable information for avoiding post-harvest anthracnose on kiwifruit.

First Report of Diaporthe eres and D. unshiuensis causing Leaf Spots on Sapindus mukorossi in China.

Sapindus mukorossi Gaertn., commonly known as soapberry, is widely cultivated as a landscaping tree in Southern China. In June 2019, a foliar disease with an incidence of ∼60% occurred on trees was observed in the soapberry germplasm repository, Jianning, Sanming, Fujian, China. The symptoms initially appeared as irregular small yellow spots, while the center of the lesions became dark brown with time. Fragments (size 3 to 4 mm2) taken from lesion margins were sterilized and cultured based on Wang et al. Two isolates (FJ1 and FJ21) were obtained with the following morphological characteristics on PDA, (1) FJ1: Conidiogenous cells were 9.7 to 25.0 × 1.5 to 2.2 μm (n=20). Alpha conidia were 6.1 to 8.3 × 2.2 to 3.0 μm (n=30), aseptate, hyaline, smooth, ellipsoidal. Beta conidia were 28.3 to 38.2 × 1.3 to 1.7 μm (n=30), hyaline, smooth, curved to hooked. Conidial drops were milky colored; (2) FJ21: Pycnidia were dark brown, 280 to 843 μm (n=30) in diam., globose, or irregular on alfalfa stems. Conidiophores were hyaline, cylindrical, smooth, and slightly tapered to the apex, 17.4 to 35.4 × 1.5 to 2.6 μm (n=20). Conidiogenous cells were 14.7 to 29.7 × 1.4 to 2.6 μm (n=20). Alpha conidia were 5.6 to 7.1 × 2.4 to 3.4 μm (n= 30), hyaline, smooth, ellipsoidal, or clavate, aseptate, biguttulate. Beta conidia not observed. Conidial drops were yellow. The morphological characteristics of FJ1 and FJ21 were similar to those of Diaporthe spp.. DNA of two isolates was extracted, and the internal transcribed spacer region (ITS) and partial sequences of translation elongation factor 1-alpha (TEF1-α), calmodulin (CAL), β-tubulin (TUB), and histone H3 (HIS) genes were amplified with primers ITS1/ITS4, EF1-728F/EF1-986R, CAL228F/CAL737R, βt2a/βt2b, and CYLH3F/H3-1b, respectively. The sequences were deposited in GenBank (accession nos. MW585608 and MW768905 to MW768908 for FJ1; MT755625 and MT776728 to MT776731 for FJ21). The BLASTn results showed that the ITS, TEF1-α, TUB, HIS, and CAL sequences of FJ1 were 100, 99, 98, 98, and 99% identical to those of D. eres (NR144923, KJ210550, KJ420799, KJ420850, and KJ434999, respectively). For FJ21, BLASTing with the same loci showed 100, 100, 100, 99, and 100% similarity with those of D. unshiuensis (MH121530, MH121572, MH121607 MH121488, and MH121448, respectively). Phylogenetic analyses with the concatenated sequences placed FJ1 and FJ21 in the clades of D. eres and D. unshiuensis, respectively. Pathogenicity tests were performed by wounding leaves of 2-year-old soapberry seedlings with a sterile needle. The leaves were inoculated with D. eres and D. unshiuensis isolates, respectively, with 10 µl of conidial suspensions (106 conidia/ml). Three plants were used for each treatment, and the leaves of each plant were inoculated. The control was treated with 10 µl of sterile water. The plants were kept in a greenhouse (RH > 80%, 25 ± 2°C). In 5 days, all inoculated leaves showed lesions similar to the field symptoms. Controls were asymptomatic. Diaporthe eres and D. unshiuensis were reisolated from the diseased leaves. No fungus was isolated from the control. Previously, D. biconispora and D. sapindicola were reported as the causal agents of soapberry, but this is the first report of D. eres and D. unshiuensis causing leaf spots on S. mukorossi in China. These data will help develop effective strategies for managing this disease.

First report of Preharvest Decay caused by Colletotrichum chrysophilum on apples in Italy (South Tyrol).

Apple (Malus × domestica Borkh.) is economically the most important fruit crop in South Tyrol (Italy). At the end of the growing season 2020, necrotic lesions and chlorosis developed on leaves and premature leaf dropping was observed on the cultivars Gala, Granny Smith and Cripps Pink(cov)/Rosy Glow(cov) in the Etsch/Adige valley. After the appearance of these symptoms, small circular brownish spots were observed on above 90 % of apples in the respective orchards. Fungal isolates were obtained from symptomatic apples by culturing small portions of fruit flesh from the lesion margin on potato dextrose agar (PDA) at 25°C in the dark. The colonies showed a white to peachy color on the upper surface and were greyish on the reverse side. Conidia were cylindrical, predominantly rounded and averaged 16.39 ± 1.37 µm and 5.75 ± 0.81 µm (n = 33), consistent morphological characteristics as described recently in Fuentes-Aragón et al. (2021). A multi-locus sequence analysis according to Astolfi et al. (2022) and Weir et al. (2022) was conducted based on the internal transcribed spacer (ITS) region and on fragments of actin (ACT), DNA-(apurinic or apyrimidinic site) lyase (APN2), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), beta-tubulin (TUB2), and intergenic spacer and partial mating type (Mat1-2) genes. MegaBlast analysis revealed 100 % identity to the reference genome of C. chrysophilum for ITS (NR_160821), for ACT (KX093982), APN2 (KX094018), CAL (KX094063), GAPDH (KX094183), 99.29 % for TUB2 (KX094285), 99.85 % for Mat1-2 (KX094325) and 99.87 % for GS (KX094204). Sequences were deposited in GenBank (ITS: OK485032; ACT: OK539650; APN2: ON624100; CAL: ON624104; GAPDH: OK539654; GS: ON624108; TUB2: OK539658; Mat1-2: ON645973). Pathogenicity assays were performed on wounded and unwounded apples of cvs. Gala and Rosy Glow(cov). Apples of both cvs. were wounded with a sterile needle. For each variant, 8 fruits were inoculated with 10 µL spore suspension (1 × 106 spores mL-1) in 0.05 % Tween®20 or with 10 µL 0.05 % Tween®20 as control. The apples were put into plastic boxes containing moist tissue and incubated at 25°C, 100 % relative humidity and 12 h photoperiod. First lesions appeared on inoculated wounded fruits after 5 days, whereas unwounded and control fruits remained asymptomatic. After 15 days, symptoms could be observed only on inoculated wounded apples with spore suspension: on 95 % of individual wounds of Rosy Glow(cov) and on 77.5 % of Gala. Koch's postulates were fulfilled by re-isolating the fungus and by identifying the re-isolates as C. chrysophilum (ITS: OK485033-OK485035; ACT: OK539651-OK539653; APN2: ON624101-ON624103; CAL: ON624105-ON624107; GAPDH: OK539655-OK539657; GS: ON624109; TUB2: OK539659-OK539661; Mat1-2: ON645974-ON645976). To date, only one study confirmed C. chrysophilum as causal agent of apple bitter rot in Europe (Cabrefiga et al. 2022). Recently, Astolfi et al. (2022) reclassified C. chrysophilum as the main causal agent of GLS on apples in Southern Brazil and Uruguay. The authors stressed that C. chrysophilum might also be the potential agent of GLS in Europe (Astolfi et al. 2022). This confirms the observations made in 2020 in South Tyrol, where massive leaf spots preceded the symptoms on fruit. To the best of our knowledge, this is the first report of a preharvest decay on apples caused by C. chrysophilum in Italy (South Tyrol).

First Report of Diaporthe eres Causing Leaf Spot of Viburnum odoratissimum var. awabuki in China.

Viburnum odoratissimum var. awabuki (K. Koch) Zabel ex Rumpl. is an evergreen tree, used as a landscape plant in China. In June 2019, a foliar disease of ~60% incidence was observed on V. odoratissimum var. awabuki at the campus of Nanjing Forestry University, Jiangsu, China. The symptoms were initially irregular small red-brown spots, later enlarged and became brown to black. Small pieces of tissue (3 to 4 mm2) cut from lesion margins were surfaced sterilized in 75% ethanol for 30 s and 1.5% NaClO for 60 s, then rinsed in sterile water and placed on potato dextrose agar (PDA) at 25℃. Pure cultures were obtained from the tip of hyphae. Using the standard phytopathological procedure, two representative isolates (SH161 and SH181) were obtained and deposited at Nanjing Forestry University. The colony on PDA was white with aerial mycelium, radiate, and the reverse was white. Black pycnidia developed on the sterilized alfalfa stems at 25°C with a 14/10 h light/dark cycle for 20 days. Conidiophores were hyaline, branched, straight to sinuous, 9.4 to 26.0 × 1.0 to 2.5 μm (n=30). Conidiogenous cells were 2.1 to 15.1 × 0.9 to 2.5 μm (n=30). Alpha conidia were 7.4 ± 0.6 × 2.0 ± 0.2 μm (n=50), hyaline, ellipsoidal to lanceolate. Beta conidia were 29.5 ± 1.8 × 1.1 ± 0.1 μm (n=30), aseptate, hyaline, smooth, curved to hooked. Morphological features of two isolates matched those of Diaporthe spp.. DNA of two isolates was extracted and the internal transcribed spacer region (ITS), partial translation elongation factor 1-alpha (TEF1-α), calmodulin (CAL), beta-tubulin (TUB), and histone H3 (HIS) genes were amplified with primers ITS1/ITS4, EF1-728F/EF1-986R, CAL228F/CAL737R, βt2a/βt2b and CYLH3F/H3-1b. The sequences were deposited into GenBank (Accession Nos. for isolate SH161: OK326730 for ITS, OK413403 to OK413406 for TUB, CAL, HIS and TEF1-α; and isolate SH181: OK331347 for ITS, OK413407 to OK413410 for TUB, CAL, HIS, and TEF1-α). BLAST search of SH161 showed high similarities with sequences of Diaporthe eres (AR5193) [KJ210529 (ITS), Identities = 438/512, (94%); KJ420850 (HIS), Identities = 466/472, (99%); KJ210550 (TEF1-α), Identities = 345/350, (99%); KJ434999 (CAL), Identities = 344/345, (99%); KJ420799 (TUB), Identities = 508/517, (98%)]. BLAST results of SH181 are listed in Supplementary Table 1. Maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and MrBayes v. 3.2.6 with the concatenated sequences placed SH161 and SH181 in the clade of D. eres. Based on the multi-locus phylogeny and morphology, two isolates were identified as D. eres. The pathogenicity was tested on 1-yr-old cuttings of V. odoratissimum var. awabuki in the greenhouse. Healthy leaves were wounded with a sterile needle, then inoculated with 5-mm plugs from the edge of two isolates cultures. The PDA plugs were used for controls. Three plants were used for each treatment, and three leaves of each plant were inoculated. Each plant was covered with a plastic bag, and sterilized water was sprayed into the bags bidaily to maintain humidity and kept in a greenhouse at the day/night temperatures at 25 ± 2°C/16 ± 2°C. Three days after inoculation, the inoculated leaves appeared lesions similar to those in the field. The controls remained healthy. Diaporthe eres was reisolated from inoculated leaves. No fungus was isolated from controls. Diaporthe eres was reported from Viburnum lantana in Austria. Also, it was reported from V. odoratissimum and V. tinus in Ukraine. This is the first report of D. eres causing V. odoratissimum var. awabuki leaf spots in China. This finding will provide an effective basis for developing control strategies for the disease.

First report of Colletotrichum siamense causing leaf spot on Michelia macclurei in China.

Michelia macclurei Dandy is an excellent timber and ornamental tree native to South China (Lan et al. 2010). In May 2020, a leaf spot disease of M. macclurei was found on the campus of Jiangxi Agricultural University (N28°45'56″, E115°50'21″). Approximately 25% (9 out of 35) of 32-year-old M. macclurei trees showed the leaf spot disease. On average, 40% of the leaves per individual tree were affected. The symptoms began as small dark brown lesions formed along the leaf margins and tips. The lesions' center was sunken with a dark brown border as the disease developed. Thirty pieces (5 × 5 mm) from the lesion margins were surface sterilized in 70% ethanol (30 s), then in 3% NaOCl (1 min), and finally rinsed three times with sterile water. Leaf pieces were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation. Sixteen strains with similar morphological characterizations were isolated, and three representative isolates (HX-1, HX-2, HX-3) were used for morphological and molecular characterization. The three isolates were white, cottony, and light gray on the reverse, producing dark-green pigmentation near the center. The conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 12.8-17.5 × 4.5-5.7 µm (14.7 ± 1.2 × 4.8 ± 0.2 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, and ranged from 5.9-8.8 × 4.4-6.7 µm (7.1 ± 0.6 × 5.6 ± 0.6 µm, n=100). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin2 (TUB2) were sequenced using the primers ITS1/ITS4 (White et al. 1990), ACT-512F/ACT-783R, CL1/CL2, CHS-79F/CHS-345R, GDF/GDR, and T1/Bt2b, respectively (Weir et al. 2012). The sequences were deposited into GenBank (Accession Nos.: MZ323328, MZ323329, MW581269 for ITS, MZ889002, MZ889003, MW661166 for ACT, MZ889004, MZ889005, MW661167 for CAL, MZ889006, MZ889007, MW661168 for CHS-1, MZ889008, MZ889009, MW661169 for GAPDH, MZ889010, MZ889011, MW661170 for TUB2). A maximum likelihood and Bayesian posterior probability-based analyses using IQ-tree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (ITS, ACT, CAL, CHS-1, GAPDH, and TUB2 ) placed three isolates in the clade of Colletotrichum siamense Prihastuti, L. Cai & K. D. Hyde. Based on the morphological characteristics and molecular data, three isolates were identified as C. siamense (Fu et al. 2019).The pathogenicity of each isolate was tested on potted 2-yr-old seedlings of M. macclurei grown in a greenhouse at 25 ℃, 70% relative humidity with a 12-h photoperiod. Twenty healthy leaves on 10 M. macclurei plants were inoculated with 10 μL of spore suspension (106 conidia/mL). Another 20 healthy leaves were inoculated with sterile water as the control. All leaves were wounded with a sterile needle (Φ=0.5 mm). The resulting symptoms were similar to those on the original infected plants, whereas the control leaves remained asymptomatic for 8 days after inoculation. C. siamense was consistently re-isolated only from the diseased leaves, fulfilling Koch's postulates. C. siamense can cause leaf diseases in a variety of hosts, such as Salix matsudana (Zhang et al. 2021), Liriodendron chinense [Hemsl.] Sarg. × tulipifera L. (Zhu et al. 2019) and Magnolia grandiflora (Zhou et al. 2022). This is the first report of C. siamense associated with leaf spot disease on M. macclurei in China, and its potential threat should be evaluated in the future. These results will help to develop effective strategies for appropriately managing this newly emerging disease.

Identification of Neofusicoccum parvum as the causative agent of leaf spot disease on Bletilla striata in China.

Bletilla striata (Thunb.) Rchb. f. (Orchidaceae) is an essential traditional Chinese medicinal plant used to treat hemorrhage, swelling, inflammation, ulcers, and pulmonary diseases (Xu et al. 2019). In April of 2020, an unknown leaf spot disease was observed on B. striata in a plantation (~ 0.2 ha) in Nanning, Guangxi province, China. Disease incidence was estimated at approximately 25% (n = 150 plants). The initial symptoms were small brown circular spots, which then expanded into reddish to brown, circular to irregular lesions 5-10 mm in diameter. As the disease developed, the whole leaf became densely covered with lesions. Finally, the lesions coalesced, killing the leaf and resulting in defoliation. To isolate the causal agent, six symptomatic leaves were collected from individual plants. Small pieces (~ 5 mm2) were cut from the margin of the necrotic lesions (n = 18), disinfected in 1% NaOCl for 2 min before rinsing three times in sterile water, and placed on potato dextrose agar (PDA) at 26°C for 3 days. Hyphal tips from the resulting cultures were transferred to PDA to obtain pure cultures. Fifteen isolates were obtained, of which twelve isolates exhibited similar morphology. Colonies on PDA were initially white, then turned dark gray after 7 days. Pycnidia were produced on the surface of PDA after 50 days. Conidia were hyaline, aseptate, ellipsoidal to fusiform, externally smooth, thin-walled, and measuring 11.5 to 15.2 × 4.9 to 6.1 μm (mean ± SD: 13.4 ± 1.0 × 5.4 ± 0.3 μm, n = 60). Morphological features were similar to N. parvum (Phillips et al. 2013). For further molecular identification, the internal transcribed spacer (ITS) region, partial translational elongation factor subunit 1-α (EF-1α), β-tubulin (TUB2) genes were amplified and sequenced using the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F (Carbone and Kohn 1999)/EF-2 (O'Donnell et al. 1998), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Sequences of the two isolates BJ-111.1 and BJ-111.4 were deposited in NCBI GenBank under the following accession numbers: OM348509-10, OM397537-40. The obtained ITS, EF1-α, and TUB2 sequences showed 99% (514/516, and 513/516 bp), 99% (275/276, and 274/275 bp), and 99% (429/431, and 429/430 bp) homology with several GenBank sequences of the ex-type strain N. parvum CMW 9081 (AY236943, AY236888, and AY236917, respectively) (Zhang et al. 2017). In addition, a phylogenetic analysis confirmed the isolates as N. parvum. Therefore, the isolates were identified as N. parvum based on morphological and molecular evidence. Furthermore, pathogenicity tests were carried out on 1.5-year-old B. striata plants. Healthy leaves on six plants (1 leaf per plant) were inoculated with a 10-μl droplet of conidial suspensions (106 conidia/mL). Three plants treated with sterile water served as the control. All plants were covered with transparent plastic bags and incubated in a greenhouse at 26°C with a 12 h photoperiod. Six days post-inoculation, the inoculated leaves showed leaf spot symptoms, while the control plants remained healthy. The experiments repeated three times showed similar results. Finally, N. parvum was consistently re-isolated from the infected leaves and confirmed by morphology and sequencing, fulfilling Koch's postulates. No fungus was isolated from the controls. To our knowledge, this is the first report of N. parvum causing leaf spot of B. striata worldwide. This result will help develop disease management strategies against this pathogen.

First Report of Top Blight of Cunninghamia lanceolata Caused by Diaporthe unshiuensis and Diaporthe hongkongensis in China.

Cunninghamia lanceolata (Lamb.) Hook. is an important conifer species widely planted in southern China. A top blight, with an incidence of 20% (40/200 seedlings), occurred on 1-year-old seedlings of C. lanceolata in a nursery, Luzhai, Guangxi, China in August 2021. The disease mainly occurred on shoot tips. The infected needles and shoots appeared brown to brownish red. White conidial tendrils oozed from pycnidia under wet-weather conditions. Lesion margins from fresh samples were cut into small pieces (n=100), which were sterilized according to Mao et al., and placed on potato dextrose agar (PDA) at 25°C. Three isolates (GXJ2, GXJ4, and GXJ6) were obtained and deposited in The China Forestry Culture Collection Center (CFCC 55717, CFCC 55716, and CFCC 55722). The colony of GXJ2 on PDA was white, with sparse aerial mycelia, and became grey with time. The α conidia were fusiform, hyaline, and aseptate, 6.7±0.6 μm × 2.6±0.2 μm (n=30). The β conidia were filiform, hyaline, and curved, 30.4±2.1 μm × 1.4±0.1 μm (n=30). Colonies of GXJ4 and GXJ6 were white, with moderate aerial mycelia, which collapsed at the center, and the collapsed parts were iron-gray. The α conidia were 7.8±0.8 μm × 2.5±0.2 μm (n=30). The β conidia were absent. Morphological characters of 3 isolates matched those of Diaporthe spp.. The partial sequences of ITS, EF1-α, CAL, β-tub, and HIS genes were amplified with primers ITS1/ITS4, EF1-728F/EF1-986R and CAL228F/CAL737R, βt2a/βt2b and CYLH3F/H3-1b according to Gomes et al. 2013, respectively. The sequences for the five genes of each of 3 isolates were deposited in GenBank (Accession Nos. see Supplementary Table 1). BLAST results showed that the ITS, EF1-α, β-tub, HIS, and CAL sequences of GXJ2 were highly similar (>99%) with sequences of Diaporthe unshiuensis, while sequences of GXJ4 and GXJ6 were highly similar (>99%) to those of D. hongkongensis (Supplementary Table 1). Phylogenetic analyses using concatenated sequences placed GXJ2 in the clade of D. unshiuensis, while GXJ4 and GXJ6 in the clade of D. hongkongensis. Based on the phylogeny and morphology, GXJ2 was identified as D. unshiuensis, GXJ4 and GXJ6 as D. hongkongensis. Pathogenicity tests were performed on nine 1-year-old seedlings of C. lanceolata, and 10 needles at shoot tip per seedling were slightly wounded and inoculated with 5-mm mycelial plugs from one of 3 isolates. Three control seedlings were treated with PDA plugs. Each plant was covered with a plastic bag after inoculation and kept in an air-conditioned nursery at 25°C/16°C (day/night). The symptoms appeared 5-8 days after inoculation and were similar to those observed in the nursery. D. unshiuensis and D. hongkongensis were re-isolated from the inoculated seedlings and were confirmed based on morphology and ITS sequences. The controls were symptomless, and no fungus was isolated from them. D. unshiuensis was first reported as an endophyte on the fruit of Citrus unshiu, and caused peach constriction canker, shoot blight of kiwifruit. D. hongkongensis was first described from fruit of Dichroa febrifuga and caused shoot canker of pear, shoot blight and leaf spot of kiwifruit, and fruit rot of peach. This is the first report of D. unshiuensis and D. hongkongensis causing the top blight of C. lanceolata. This study provides a basis for controlling this newly emerging disease in the nursery.