Black Lesions Research Articles

Pudrición del tallo (Sclerotium oryzae) en cultivos de arroz en la región sur de Paraguay

The expansion of rice cultivation in Paraguay observed in the last five agricultural seasons, together with the new technologies applied, has been accompanied by an increase in the presence of diseases affecting rice leaf sheaths and stems. In view of the scarcity of information in the country on some of these diseases, this study was carried out to characterise rice stem rot. Samples of symptomatic plants were collected from commercial lots located in the southern region of the country, and routine techniques in plant pathology for the study of mycoses were applied, such as macro and microscopic observations, in vitro sowings, and the study of morphological and cultural characteristics of the fungus. The pathogenicity of the fungus was determined by inoculations on healthy 50-day-old rice plants. Associated with the symptoms observed on rice pods and stems, the fungus Sclerotium oryzae Catt was identified as causing irregular black lesions on the leaf sheaths at the water line. As the disease progressed, it caused lesions on the sheaths and leaf blades, and finally stem rot with the formation of sclerotia inside the affected stems. The symptomatology observed in the rice plants and the cultural and morphometric characteristics of the pathogen Sclerotium oryzae were consistent with those reported in the bibliographical references analysed.

First Report of Leaf Black Spot Caused by Alternaria alternata on Rosemary (Rosmarinus officinalis L.) in China.

Rosemary (Rosmarinus officinalis L.) is an aromatic, evergreen, medicinally important shrub and widely used for cooking, tea, cosmetics as well as medicinal materials. It is grown in many countries including China that had more than 9300 hm2 of commercial cultivation area in 2021. In March 2020, a leaf spot disease sporadic occurred in field rosemarry plants in Nanyang City (32º51´ N, 111º36´ E), Henan Province, China. The disease outbreaked in September with a disease incidence of 57-83%. Symptoms initially appeared as small brown leaf spots that gradually expanded into dark blackbrown irregular lesions. Most of the spots started from the leaf tip or leaf margin, and gradually spread to the leaf base, resulting in heavy defoliation especially on rainy days. Diseased leaf segments (1×3 mm) were surface-sterilized by dipping in 1% sodium hypochlorite for 1 min, rinsed three times with sterile distilled water, and plated on potato dextrose agar, then incubated at 28°C in the dark for 5 days. Twelve fungal isolates with the same morphological characteristics were obtained from nine affected leaves. The fungal colonies were initially white and turned gray brown with flocculent aerial mycelia and a whorled back. Conidia were frequently born in a long chain, with a short beak, brown or light-brown, 13.2 to 48. 7 (average 26.1) × 4.0 to 13.1 (average 8.0) μm in size (n=148) with 0 to 8 transverse and 0 to 3 longitudinal/oblique septa. Phenotypic features of the isolates agreed with those of Alternaria alternata (Simmons et al. 2007). Two isolates Aa1 and Aa2 were randomly selected for molecular and pathogenicity tests. DNA was extracted from mycelia. Partial sequences of internal transcribed spacer (ITS) and translation elongation factor 1-alpha (TEF1-α) were amplified using the primer pairs ITS1/ITS4 and EFI-728F/EFI-986R (Wei et al. 2022), respectively. The GenBank accession nos. were OK036714 and OK036715 for ITS, and ON951980 and ON951981 for TEF1-α of Aa1 and Aa2, respectively, with a maximal identity of greater than 99% to multiple A. alternata strains. In the neighbour joining phylogenetic tree of the amplified ITS and TEF1-α sequences both Aa1 and Aa2 clustered with A. alternata strains, clearly separating them from other Alternaria spp. For pathogenicity test, conidial suspensions (1×106 spores /mL) of Aa1 and Aa2 were separately sprayed on healthy one-year-old rosemary plants (n=3) with their leaves slightly wounded with a sterilized needle. Control plants (n=3) were sprayed with sterile water. Both inoculated and control plants were incubated at 90% RH, 28 °C. After 14 days, all the inoculated leaves showed black brown lesions similar to those on naturally affected field plants, whereas controls remained symptomless. Fungal cultures with the same phenotypic features as the inocula were constantly re-isolated from the infected leaves. A. alternata was reported as pathogen causing foliar necrosis on rosemary in Italy (Perello et al.1995) and leaf spot (or leaf blight) on multiple plant species such as Actaea dahurica (Hai et al. 2022), and Ligustrum japonicum (Wei et al. 2022) in China. This is the first report of A. alternata causing leaf black spot on rosemary in China.

First report of leaf spot disease caused by Fusarium oxysporum on Litsea cubeba in China.

Litsea cubeba, an important industrial plant species that originated in China, produces fruit essential oil extensively applied in the chemical industry (Xiang et al. 2020). In July 2020, a large-scale outbreak of leaf spot disease on Litsea cubeba was first observed and then monitored over time in Yueyang (29°37'N; 113°13'E) and Changsha (28°06'N; 113°02'E), Hunan province, China. Symptoms of this disease consisted of round-shaped lesions that initially appeared as small light-brown spots. With the increase in number, these small spots coalesced into larger, dark-brown lesions leading to yellowing and abscission of the leaves. To identify the causal agent this disease, the pathogen was isolated with a tissue separation method (Gao et al. 2020). The infected leaf tissues surface-disinfected with 75% ethanol and 0.1% HgCl were aseptically cut into small pieces (11 cm) and then placed onto potato dextrose agar (PDA) medium with cephalothin (0.2 mg/ml) and incubated at 28°C for 3-5 days. The purified colonies on PDA exhibited fluffy white hyphae, secreted a dark red pigment that had been observed in previous studies (Xiao et al. 2015) and produced microconidia and macroconidia. The microconidia were single-celled, non-septate, ovoid, and ranged from 3.08 to 13.89 μm long and 2.17 to 3.62 μm wide (n=50). Macroconidia were three to five-septate, slightly curved, and ranged from 11.77 to 26.85 μm long and 3.31 to 4.50 μm wide (n=50). These morphological features suggested that theisolates were most likely Fusarium oxysporum (Savian et al. 2021). To further confirm the identity of this pathogen (designated as Fox-1), the TEF-1a gene (Genbank accession No. OM281065) and rDNA ITS region (Genbank accession No. OM250084) were cloned and then sequenced (Cui et al, 2021). Sequence alignments indicated that the ITS and TEF-1a sequences shared 99.8% (504/505) and 99.7% (665/667) similarities with that of F. oxysporum (Genbank accession No. MF667966, KT230848), respectively. Both of the morphological characteristics and molecular data were used to identify this pathogen as F. oxysporum Schltdl.: Fr. 1824. To further verify whether these isolates of F. oxysporum can cause leaf spot disease, Koch's postulates were tested (Gradmann 2014). The purified pathogens were inoculated on artificial wounds of detached Litsea cubeba leaves and the leaves on the field plants of Litsea cubeba, respectively. The wounds of leaves were inoculated with sterile distilled water as negative controls. The experiment was performed independently three times, each with three leaves and three inoculated wounds on each leaf. All pathogen-inoculated wounds developed dark brown or black lesions on detached leaves within 3 days and on leaves on plants within 9 days, whereas the controls showed no symptoms. Re-isolations from infected leaves confirmed that the re-isolated pathogens possessed identical morphological characteristics to those of the original pathogens. To our knowledge, this is the first report of leaf spot infection of Litsea cubeba caused by F. oxysporum in China. This disease severely delays plant development and significantly decreases the yield of essential oil of Litsea cubeba. Our results laid a foundation for the subsequent research into pathogenic mechanisms drug sensitivity tests, which will contribute to the prevention and cure of leaf spot disease of Litsea cubeba.

Creeping hair: A case report

Introduction: Creeping hair is a rare condition that presents as a migratory linear cutaneous track and accounts for 1% of all subtypes of creeping eruption. Herein, we report a case of creeping hair on the neck that was diagnosed by histopathological examination. Case presentation: A 32-year-old man presented to our outpatient clinic with a half-day history of a visible black linear lesion in the left neck region without any symptoms. He had no recent travel history and no special personal or family medical histories. Dermatological examination revealed a black, thin, linear, 2-cm-long skin lesion in the left neck region, leaving a pattern similar to wave-like erythema or cutaneous larva migrans. Histopathological examination of a skin biopsy confirmed the diagnosis of creeping hair. The treatment was a shallow incision over the lesion and removal of the hair. Discussion: There are few reports of creeping hair, and the pathogenesis remains unknown. The lack of a hair follicle in the dermis on histopathological examination suggests that the creeping hair in the present case was an outgrown hair that penetrated the dermis under constant friction and extrusion. Conclusion: Fragile skin and constant friction are common reasons for creeping hair. The creeping hair in this case was classified as an outgrown hair.

First Report of Colletotrichum nymphaeae CausingWalnut Anthracnose in China.

Walnut(Juglans regiaL.) is a high quality woody nut and edible oil tree with a planting area of about 5,000,000 hectare in China. Walnut anthracnose is a serious disease, infecting approximately 50% of the fruits and causing a great yield losses (Wang et al. 2016). In 2019 to 2020, walnutfruits with anthracnose symptoms were collected from walnut orchards in province of Hubei, Sichuan procinve and Chongqing municipality, China. Symptoms on fruits were circular or subcircularor irregular shaped, with brown to black water soaked and sunken lesions. The black lesions enlarged and amalgamated into large necrotic areas. The older spots in the center became blackish with acervuli causing the complete mummification of the fruit, and orange conidial masses appeared under wet conditions. Necrotic tissues of the fruits were sterilized in 75% ethanol solution for 30 s, then sterilized in 4% sodium hypochlorite for 1min, and washed 3 times with sterile distilled water. The tissues were put on potato dextrose agar (PDA) and incubated at 25℃. Pure cultures were obtained by single-spore culturing method and the isolate HBBK4-4 was deposited into the China's Forestry Culture Collection Center (CFCC 57388). On PDA, the colonies were cottony, white to pale gray with aerial mycelium on the upper side and pink with black spots on the reverse. The mycelial growth rate was 9.6 mm/day at 25°C. Conidia were 1-celled, colorless, smooth-walled, straight, cylindrical to cylindrical-clavate with acute ends, 12.5 to 18.2 × 3.9 to 5.4 μm (mean 15.3 ± 3.7 × 4.9 ± 0.6 μm,n= 40). Most conidia germinated and developed one pleurogenous, 1-celled appressorium. Appressoria were single, medium brown, smooth-walled, ovate to ellipsoid, 5.4 to 7.8 × 5.4 to 7.8 μm (mean 6.7 ± 0.6 × 6.3 ± 0.5 μm,n= 30). These morphological characteristics were in concordance with published descriptions of Collectotrichum acutatum species complex. To further confirm the identity, internal transcribed spacer (ITS), beta-tubulin (TUB2), chitin synthase 1 (CHS-I) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified and sequenced (Damm et al. 2012). The ITS (OM189549) and TUB2 (OM273642) sequences of isolate HBBK4-4 showed 100% similarity, and GAPDH (OM249791) and CHS-1 (OM273641) sequences showed 98.7% and 99.6% similarity withC. nymphaeaeCBS100064 respectively. A maximum likelihood phylogenetic tree was generated based on combining all sequenced loci in MEGA5. 18 isolates including HBBK4-4 fell in theC. nymphaeaeclade with 96% bootstrap support. To verify Koch's postulates, six isolates were used for pathogenicity test, and 20 healthy fruits and 15 fully expanded leaves for each isolate were inoculated with 5-mm-diameter mycelial plugs. Controls consisted of detached premature fruits inoculated with a PDA plug without the fungus. Six days after inoculation, all fruits and leaves developed anthracnose symptoms similar to those observed in the field, while the controls remained healthy. The pathogenicity tests were repeated twice with the same results. The morphology of the reisolated fungi was consistent with the inoculated one, fulfilling Koch's postulates.The isolate HBBK4-4 was identified as C. nymphaeae, based on the description byDamm et al. (2012). The species C. nymphaeae has been previously reported to cause severe anthracnose on walnut in France (Da Lio et al., 2018), Brazil (Savian et al., 2019) and Italy (Luongo et al., 2022). To our knowledge, this is the first report ofC. nymphaeaeas a pathogen ofwalnutanthracnose in China. The result provided crucial information for epidemiologic studies and management of this disease.

First report of Coniella granati causing leaf spot of pomegranate (Punica granatum L.) in Hungary.

Pomegranate (Punica granatum L.), the hystoric fruit and ornamental crop native to Iran and North India is widely planted in the Mediterranean and became popular in the house gardens of northest parts of Europe (Fernandez et al. 2014) including Hungary. In August 2020 necrotic black lesions and serious defoliation were observed on 60% of 1-3 year old pomegranate trees (cv. Wonderful) in a horticultural nursery near Gödöllő, Hungary (47°36'00.9"N 19°21'26.5"E). Symptoms started as small irregular dark brown spots on the leaves, which later increased in size (2.6 ± 0.9 mm). Ultimately, the entire leaf turned yellow, defoliation resulted in damage on (6) - 8 - (15)% of the leaves. Then, black pycnidia with unicelled, elliptical to fusiform, colourless conidia (Avg. 50 conidia: 2.4 - (3.6) - 3.9 × 10.2 - (13,1) - 17.9 µm) developed on the surface. These morphological features matched those described earlier by Van Niekerk et al. (2004) and Alvarez et al. (2016) for C. granati. Conidia from pycnidia were directly transferred to potato dextrose agar (PDA) by sterile needle. The plates were incubated at 24°C in the dark. Light yellow colonies with whitish aerial mycelia and later black globose pycnidia were observed. Mass of conidia oozed from pycnidia after 15 days of incubation. Pathogenicity tests were carried out on 1-year-old potted P. granatum trees (cv. Wonderful) with 5 replicates in the greenhouse. Ten, randomly selected leaves were inoculated per plant. 7-mm mycelial plugs from the edge of 10-day-old colonies were placed directly on disinfested (2% NaOCl solution, than sterile distilled water) leaves. The plants were covered with plastic film for 3 days after inoculation (26±3°C and 87±3% relative humidity). Pathogenicity was also tested on nonwounded, surface-disinfested fruits by mycelial plugs in 3 × 3 replicates. Inoculated fruits were placed in large grass vessels for 15 days (24±2°C and 80±5% relative humidity). Uncolonized, sterile PDA plugs were used as controls in both cases. Dark brown legions developed after 9-12 days on the plants in the greenhouse. On pomegranate fruits, the fungus colonized the fruit after 7-8 days, followed by fruit rot. In some cases, after 2 weeks pycnidia developed on the skin surface. No decay were present on control leaves or fruits. The pathogen was reisolated from all infected tissues and identified as C. granati, thus fulfilling Koch's postulates. For molecular identification, total genomic DNA of the isolate was extracted from the growing margins of colonies on PDA and partial sequence of internal transcribed spacer (ITS) and translation elongation factor 1-alpha (tef1) were amplified by PCR using primers described by Alvarez et al. (2016). Sequence data of the Hungarian isolate of the ITS region (GenBank acc. no. MW581953) showed 99.8% identity (559 bp out of 560 bp) with C. granati sequences deposited in GeneBank (Acc. nos. MH860368, MH855389 and KX833582). Considering tef1 sequence of the Hungarian isolate (OM908764) obtained had complete identity with other published C. granati isolates (KX833676, KX833682). C. granati has been previously reported on pomegranate from Europe (Palou et al. 2010, Pollastro et al. 2016). Based on morphological and molecular studies, this is the first record of C. granati in Hungary. The economic importance of this disease in currently limited in Hungary due to pomegranate is rather an ornamental crop, however, the first cultivation trials have been already started. There is a risk that the spread of the pathogen began with the infected propagating material, as a result the disease may outbreak anywhere in the country.

First report of Colletotrichum siamense associated with anthracnose on Theobroma cacao fruits in Venezuela

Cacao (Theobroma cacao) plantations are spread throughout Venezuela, but the cultivar “Sweet cacao” or “Criollo (= Creole)” is concentrated in the states of Mérida, Táchira, and Zulia (Palma, 1953). In April 2016, brown and black sunken lesions typical of anthracnose symptoms were observed on cacao fruits in plantations from the municipalities Alberto Adriani, Andrés Bello, Antonio Pinto Salinas, Obispo Ramos de Lora, and Sucre in Mérida State, Venezuela (Figure 1). For isolation of the pathogen, fruits were cut into c. 5 mm2 sections at the healthy and diseased margin. Tissues were then surface disinfected for 30 seconds in 70% ethanol, 60 seconds in 0.5% sodium hypochlorite, washed three times with sterilised water for 60 seconds, and dried with sterilised filter paper. Disinfected samples were plated onto 2% malt extract agar and incubated at 25°C for seven days or until mycelia were observed growing from the samples. The fungal mycelium was sub-cultured onto potato dextrose agar (PDA), and hyphal tipping methods were performed to obtain pure cultures that were used for morphological analysis. Nine strains were isolated and evaluated. Colonies on PDA grew to 60–80 mm (mean = 70), in diameter in seven days, and >90 mm diameter in 10 days at 25°C. After 15 days, colonies were flat with an entire edge, with aerial mycelium that were dense, cottony, pale olivaceous grey on the surface and buff to pale luteous on the underside of the culture (Figure 2). Conidiomata were acervular with conidiophores formed on a cushion of roundish and medium olive-brown cells; setae were not observed. Conidiogenous cells were hyaline, cylindrical to ampulliform, simple, short, straight, measuring 9–14 × 1.1–1.2 µm. Conidial mass orange in colour and visible through the mycelium in the reverse of the culture. Conidia were hyaline, smooth-walled, cylindrical, aseptate, with ends rounded and often tapering slightly from the middle towards the base or only sometimes narrowed at the centre (sub-cylindrical), 8–13 × 2.4–3.8 µm, mean ± SD = 10.1 ± 1.4 × 3 ± 0.3 µm, L/W ratio = 3.4 (Figure 3). Appressoria measuring 5–9 µm diameter, terminal, subglobose or with one or two borders lobed, brown, solitary were observed (Figure 4). No chlamydospores were observed. The morphological characteristics of these isolates placed this fungus within the Colletotrichum gloeosporioides species complex (Weir et al., 2012). The glutamine synthetase (GS) and Apn2-Mat1-2 intergenic spacer and partial mating type (Mat1-2) gene (ApMat) were amplified using the primer pairs GSLF2/GSLR1 (Liu et al., 2016) and AMF1/AMR1 (Silva et al., 2012), respectively. The ApMat sequences were deposited in GenBank (Accession Nos. MF436054-MF436062) and GS sequences (MF436063-MF436071). Maximum likelihood (ML) was evaluated with a bootstrapping method and Bayesian inference (BI) analysis was performed with MrBayes v. 3.2.6 phylogenies, both calculated in Geneious 2021.0.3.0. The posterior probabilities from ML/BI analyses (87/0.99) in the combined dataset supported the inclusion of T. cacao isolates within the Colletotrichum siamense clade (Figure 5). Therefore, using phylogenetic analyses based on sequence data from the two loci (GS and ApMat) and morphological characteristics of the fungus, we identified the isolates as C. siamense. ApMAT and GS loci provided robust support to separate all Colletotrichum spp. inside the C. gloeosporioides complex species, confirming that these loci provide higher phylogenetic resolution compared to other standard markers (Silva et al., 2012; Liu et al., 2015). Colletotrichum gloeosporioides has been reported to cause anthracnose on leaves, flowers and unripe cacao fruits in Venezuela (Reyes & Capriles de Reyes, 2000), and was also present at high incidence on fruits in the municipality of Sucre, Merida state (Parra et al., 2009). Morphological similarities between C. gloeosporioides and C. siamense, such as the orange colour of conidial masses that developed in cultures, could have led to misidentification of the causal agent of the disease previously in Venezuela. To our knowledge, this is the first report of C. siamense associated with anthracnose on cacao fruits in Venezuela. We are grateful to the U. S. Department of State Fulbright Scholar Program for supporting a one-year exchange visit and providing financial support to Prof. Mohali Castillo, ID assigned: PS00236102, as researcher in the Department Agricultural Biology, Colorado State University. Further, we thank Kristen Otto for excellent laboratory assistance.

First Report of Phyllosticta capitalensis Causing Black Spot on Radermachera hainanensis Merr. in China.

Radermachera hainanensis Merr. plants are native in south-central and southeast of China. Plants produce large flowers, and are widely cultivated in China as ornamentals. In April 2020, R. hainanensis Merr. plants grown in Cixi Lvpin Garden (30°26'54″N, 121°25'48″E), Zhejiang Province, were found to have many black circular necrotic lesions. In the early infection stage, the lesions appeared in lower leaves as small black circular spots which developed later into large spots (11 to 38 mm diameter) with grey centers and chlorotic edges. Ultimately, the spots spread and merged. Moreover, infected leaves showed premature leaf fall. Disease intensity reached approximately 20% of plants in the affected field (0.5 ha). After effective chemical control, this disease did not spread to other healthy plants in the same garden. To identify the causative pathogen associated with the disease, ten symptomatic leaves were collected from ten different plants. Leaf tissues were cut from the lesion margins and sterilized as follows: surface sterilized with 75% ethanol for 30 seconds and washed three times in sterile distilled water. The leaf tissues were then dipped into 10% sodium hypochlorite for 3-4 minutes, then washed three times in distilled water and dried on a sterile filter paper. After drying, the surface-sterilized leaf discs were cut to small pieces (3×3 mm) and transferred to potato dextrose agar (PDA) plates and incubated at 28°C for 2 to 3 days under 12 h photoperiod. A total of 15 isolates were obtained from the affected leaves, and all the isolates displayed the same colony characteristics. Then, three single-spore isolates were randomly selected (F2, F5 and F8) for further study. The fungal colonies were dark green with a granular surface, and irregular white edges, later turning black. Conidia were one-celled, oval, and narrow at the end with a single apical end, measuring from 7.8 to 11.1 × 4.6 to 5.9 μm (av. 9.5 × 5.2 μm, n=50). These morphological characteristics were consistent with the description of Phyllosticta capitalensis (Wikee et al. 2013; Guarnaccia et al. 2017). The identity of three representative isolates were confirmed by a multilocus approach. The DNA of three isolates were extracted and partial sequences of ribosomal internal transcribed spacer (ITS), actin (ACT), and translation elongation factor 1-alpha (TEF1-α) were amplified and sequenced as previously described (White et al. 1990; O'Donnell et al. 1998; Carbone & Kohn et al. 1999). The three selected isolates shared 100% identical sequence of ITS, ACT and TEF1-α. Then representative isolate F8 was selected for further study. BLAST analysis in GenBank showed that the obtained sequence of ITS (MZ317550) had 99% identity to P. elongata isolate eSX25240811. Other two sequences of ACT (MZ326837) and TEF1-α(MZ326839) showed 99% and 98% identity to P. capitalensis isolate YLWB01, respectively. The phylogenetic trees were constructed by Bootstrap method with 1000 replications using Maximum Likelihood model implemented in the MEGA 7. Results showed that the isolate F8 clustered with P. capitalensis with 100% bootstrap support. Pathogenicity of strain F8 was tested by Koch's postulates. A pathogenicity test was performed in a greenhouse with 80% relative humidity at 28°C. 20 healthy plants were sprayed with a 1×106 conidia ml-1 suspension (three leaves from each individual plants) and another 20 healthy plants were sprayed with sterile distilled water (three leaves from each individual plant) as control. Conidia was obtained from PDA plates after 7 days of incubation in the biochemical incubator at 28°C and concentration was counted in hemacytometer. After 15 days, disease symptoms were observed on all inoculated leaves, whereas the control plants remained asymptomatic. After that, P. capitalensis was re-isolated only from the infected leaves and identified by morphological and sequence analyses. Early identification of P. capitalensis as a causal agent for black spot is crucial to employ effective disease management strategies to control disease in the field. P. capitalensis has been reported on many crops in China (Cheng et al. 2019; Tang et al. 2020; Liao et al. 2020). However, to our knowledge, this is the first report of black spot disease caused by P. capitalensis on Radermachera hainanensis Merr. in China.

Clinical practice guidelines for endometriosis in Japan (The 3rd edition).

Clinical practice guidelines for endometriosis in Japan (The 3rd edition).

Biological and Molecular Characterization of the Lytic Bacteriophage SoKa against Pseudomonas syringae pv. syringae, Causal Agent of Citrus Blast and Black Pit in Tunisia

Pseudomonas syringae pv. syringae (Pss), the causal agent of citrus blast and black pit lesion of lemon fruit, continues to cause serious damage in citrus production in Tunisia. Faced with the rapid emergence of the disease and the inefficiency of conventional control methods, an alternative strategy based on the use of bacteriophages was pursued in this study. The lytic Pss bacteriophage SoKa was isolated from soil collected from Tunisian citrus orchards. Analysis of the host range showed that SoKa was able to lyse seven other Pss strains. Interestingly, Pseudomonas syringae pv. porri, pathogenic to leek, could also be infected by SoKa. The activity of SoKa was maintained at pH values between 2 and 10, at temperatures between −80 and 37 °C; the phage could resist UV radiation at an intensity of 320 nm up to 40 min. Whole genome sequencing revealed that the Pseudomonas phage SoKa is a novel phage that belongs to the Bifseptvirus genus of the Autographiviridae family. The absence of virulence proteins and lysogeny-associated proteins encoded on the phage genome, its anti-biofilm activity, and the significant reduction of tissue necrosis in different fruit bioassays make SoKa potentially suitable for use in phage biocontrol.

A Pan-Global Study of Bacterial Leaf Spot of Chilli Caused by Xanthomonas spp.

Bacterial Leaf Spot (BLS) is a serious bacterial disease of chilli (Capsicum spp.) caused by at least four different Xanthomonas biotypes: X. euvesicatoria pv. euvesicatoria, X. euvesicatoria pv. perforans, X. hortorum pv. gardneri, and X. vesicatoria. Symptoms include black lesions and yellow halos on the leaves and fruits, resulting in reports of up to 66% losses due to unsalable and damaged fruits. BLS pathogens are widely distributed in tropical and subtropical regions. Xanthomonas is able to survive in seeds and crop residues for short periods, leading to the infections in subsequent crops. The pathogen can be detected using several techniques, but largely via a combination of traditional and molecular approaches. Conventional detection is based on microscopic and culture observations, while a suite of Polymerase Chain Reaction (PCR) and Loop-Mediated Isothermal Amplification (LAMP) assays are available. Management of BLS is challenging due to the broad genetic diversity of the pathogens, a lack of resilient host resistance, and poor efficacy of chemical control. Some biological control agents have been reported, including bacteriophage deployment. Incorporating stable host resistance is a critical component in ongoing integrated management for BLS. This paper reviews the current status of BLS of chilli, including its distribution, pathogen profiles, diagnostic options, disease management, and the pursuit of plant resistance.

First Report of Leaf Spot Caused by Colletotrichum citricola on Cavendish bananas in China.

Cavendish banana (Musa spp. AAA group) is an important tropical and subtropical fruit with significant economic value. It is widely planted in Guangxi, Yunnan, Hainan, Fujian and Guangdong provinces in China. In November 2020, leaf spots were observed on nearly 80% of the plants growing in three Cavendish banana plantations in Chongzuo, Guangxi, China. The symptoms on Cavendish banana leaves initially appeared as small black necrosis spots, which gradually expanded and connected, eventually covered the entire leaf. Three diseased leaves from three plantations were collected, sectioned into small pieces (5 ×5 mm), surface sterilized (10 s in 75% ethanol, followed by 1 min in 1% sodium hypochlorite and rinsed three times in sterile water) and placed on potato dextrose agar (PDA) at 28℃ for 5 days for pathogen isolation. The fungal colonies were white, cottony, while the reverse sides were white, concentric circles with yellowish-brown discoloration in 7-day cultures. The conidia were hyaline, aseptate, cylindrical, oval, measuring 10.3 to 17.71 μm (mean 14.06 ± 1.45 μm; n = 200) in length and 4.48 to 9.57 μm (mean 7.46 ± 0.69 μm; n = 200) in width. Three representative isolates (DX1-5, LZ4-5, and FS1-3) were obtained by monosporic isolation. The partial internal transcribed spacer (ITS) regions, actin (ACT), chitin synthase (CHS-1), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), and β-tubulin (TUB2) were amplified from genomic DNA for the three isolates (Weir et al. 2012). The sequences of the amplified fragments were deposited in GenBank (accessions OL361844 to OL361858, for GAPDH, CAL, ACT, CHS-1, and TUB2 of isolate DX1-5, LZ4-5 and FS1-3; OL305066 to OL305068 for ITS) and showed over 99% identities with the corresponding sequences of C. citricola. A neighbor-joining phylogenetic tree based on the above six genes of type or ex-type specimens of Colletotrichum (Fu et al. 2019) was constructed with MEGA 5.2 using the concatenation of multiple sequences (Kumar et al. 2016). All three isolates clustered together with the type culture of C. citricola (CBS 134228, CBS 134229, CBS 134230) with 82% bootstrap support in the phylogenetic tree. According to the molecular and morphological characteristics, all three isolates were identified as C. citricola. Pathogenicity tests were conducted on one-month-old primary hardened tissue culture plantlets. Tender, healthy leaves were gently scratched with a sterile needle, and each wound site was inoculated with sterile cotton impregnated with conidial suspension (106 spores/ml) for each isolate. Wounded leaves were treated with sterile cotton impregnated with conidial suspension of C. fructicola as positive controls and sterile water as negative controls. Each isolate was inoculated with three tissue culture plantlets, six inoculated sites on each plantlet, the same as controls. All inoculated tissue culture plantlets were covered with plastic bags to maintain high humidity and placed in a 28℃ growth chamber with constant light. Black necrotic lesions were clearly observed on the inoculated leaves and the positive controls after 7 days, whereas no symptoms appeared on the negative control leaves. The fungus was re-isolated from inoculated leaves, and these isolates matched the morphological and molecular characteristics of the original isolates confirming Koch's postulates. To our knowledge, this is the first report of leaf spot caused by C. citricola on Cavendish banana worldwide.

Pathogenicity of three Botryosphaeriaceae fungi, Diplodia scrobiculata, Diplodia mutila, and Dothiorella californica, isolated from coast redwood (Sequoia sempervirens) in California

AbstractThree Botryosphaeriaceae species, not previously recorded on coast redwood, were detected from native redwood stands in Humboldt Co., CA during general surveys between 2014 and 2017. Pathogenicity experiments with three of these fungi, Diplodia scrobiculata, Diplodia mutila, and Dothiorella californica, were performed on 2‐year‐old redwood seedlings to fulfil Koch's postulates and prove pathogenicity. Only D. scrobiculata produced lesions significantly longer than control lesions, although they were generally smaller than those of more virulent Botryosphaeriaceae pathogens in previous studies. In a separate pathogenicity study of D. scrobiculata on younger seedlings without secondary growth, inoculations on wounded stems resulted in black lesions that were much more externally obvious than the wound inoculations on lignified stems after 90 days. However, no lesions developed on non‐wounded stems of younger seedlings. Lesion lengths caused by D. scrobiculata were consistent between the two studies. On one wound‐inoculated, non‐woody seedling, D. scrobiculata was recovered from asymptomatic stem tissue 2 cm below the inoculation site, indicating possible pathogen latency. These experiments indicate that there are more Botryosphaeriaceae fungi involved in coast redwood dieback than previously known and confirm the pathogenicity of D. scrobiculata on primary and secondary tissue of coast redwood.

Identification of New Fusarium sulawense Strains Causing Soybean Pod Blight in China and Their Control Using Carbendazim, Dipicolinic Acid and Kojic Acid.

Soybean plants are highly susceptible to Fusarium species, which significantly reduce soybean production and quality. Several Fusarium species have been reported to synthesize mycotoxins, such as trichothecene, which have been related to major human diseases. In November 2021, soybean pods in Nantong municipality, China, showed black necrotic lesions during the harvest stage. The disease incidence reached 69%. The pathogen was identified as Fusarium sulawense via morphological analysis and sequencing of ITS, EF1-α and RPB2 genes. A PCR assay with primers targeting the trichothecene biosynthesis genes suggested that the three isolates could synthesize trichothecenes. The effectiveness of fungicide carbendazim and natural metabolites dipicolinic acid and kojic acid was screened for the management of F. sulawense on postharvest soybean pods. The highest efficacy was obtained when combining 3.8 mg/mL carbendazim and 0.84 mg/mL dipicolinic acid (curative efficacy: 49.1% lesion length inhibition; preventive efficacy: 82.7% lesion length inhibition), or 1.9 mg/mL carbendazim and 0.71 mg/mL kojic acid (preventive efficacy: 84.9% lesion length inhibition). Collectively, this report will lead to a better understanding of the safety hazards found in soybean products in China and reveals the application of dipicolinic and kojic acids to reduce the use of carbendazim.

Background selection schema on deep learning-based classification of dermatological disease

Skin diseases are one of the most common ailments affecting humans. Artificial intelligence based on deep learning can significantly improve the efficiency of identifying skin disorders and alleviate the scarcity of medical resources. However, the distribution of background information in dermatological datasets is imbalanced, causing generalized deep learning models to perform poorly in skin disease classification. We propose a deep learning schema that combines data preprocessing, data augmentation, and residual networks to study the influence of color-based background selection on a deep model’s capacity to learn foreground lesion subject attributes in a skin disease classification problem. First, clinical photographs are annotated by dermatologists, and then the original background information is masked with unique colors to generate several subsets with distinct background colors. Sample-balanced training and test sets are generated using random over/undersampling and data augmentation techniques. Finally, the deep learning networks are independently trained on diverse subsets of backdrop colors to compare the performance of classifiers based on different background information. Extensive experiments demonstrate that color-based background information significantly affects the classification of skin diseases and that classifiers trained on the green subset achieve state-of-the-art performance for classifying black and red skin lesions.

ORAL MELANOTIC MACULA: CASE REPORT

The oral melanotic macula is a color change of the mucosa by the focal increase in melanin and melanocytes. The objective of this work is to report the case of a 46-year-old female patient, with an 8-month evolution of a black lesion in the labial region. The patient presented to the stomatology service complaining about "a stain on her lip." On extraoral examination, a single brown macula, uniformly pigmented in the vermillion of the lower lip, noninfiltrating, smooth surface, and measuring 1.4 × 0.9 cm, was observed. On intraoral examination, there was nothing worth noting. The clinical diagnosis was a labial melanotic macula. An excisional biopsy was performed and the histologic sections stained in hematoxylin and eosin revealed areas of pigmentary incontinence of probable inflammatory cause, confirming the oral melanotic macula diagnosis. It is important that pigmented lesions are biopsied to eliminate the possibility of malignant lesion.

First Report of Fruit Rot of Sweet Pepper Caused by Cladosporium cladosporioides in Israel.

Sweet pepper (Capsicum annuum L.) is an important vegetable crop grown in Israel for local and export markets. Bell pepper 'Dinamo' and sweet bite pepper exhibiting fruit spotting were observed in postharvest storage of commercial farms in Giv'at Ko'ah (32°02'21″ N; 34°56'41″ E) and Arava (30°46'41″ N; 35°14'28″ E) in July, 2021. Fruit with lesions were sporadic at each facility, however, limited numbers of packaged boxes were discarded due to a high percentage of symptomatic fruit. The spots appeared as slightly sunken dark brown or black lesions, coated by a fine velvety layer of olive-grey conidia (Suppl. Fig. 1). A fungus was routinely isolated from the fruit onto potato dextrose agar amended with 50 ppm chloramphenicol (PDAC) and was tentatively identified as a Cladosporium spp. based on colony morphology and conidial appearance (Suppl. Fig. 1). Isolates (n = 6) of the fungus were cultured on malt extract agar (MEA) and PDA for morphological observation. On MEA, the pathogen sporulated profusely and conidia measured 4.5 (3 - 7) × 3.5 (2 - 4) µm and were smooth and globose or sub-globose. Ramoconidia had 0 or 1 septations and measured 13.4 (10 - 18) × 3.9 (3 - 6) µm. Conidiophores were solitary, straight or flexous, usually unbranched, and measured 60 - 350 µm. Both macro- and micronematous conidiophores were present. Hyphae were flat and immersed, olivaceous-grey, septate and rope-like. The fungus was slow growing, reaching 41.0 mm after 10 d incubation at 20°C. To confirm species identity, the isolates were grown in potato dextrose broth and DNA was extracted from the mycelium using MasterPure DNA Purification Kit (EpiCentre, Madison, WI, USA) following the manufacturer's instructions. The internal transcribed spacer (ITS) region was amplified using primers ITS 1 and ITS 4, and elongation factor-1 and actin gene regions with EF1-728F/986R and ACT-512F/783R, respectively (Carbone and Kohn, 1999). The PCR products were sequenced and a BLASTn search showed 99-100% similarity to Cladosporium cladosporioides (GenBank accession nos. MW255614.1, MK416093.1, MK306457.1), a common fungal species found worldwide as a plant pathogen and saphrophyte (Bensch et al., 2012). The ITS gene sequences for a representative isolate from each location were deposited in GenBank (accession nos. OK104139.1 and OL672241.1). To confirm pathogenicity, red bell pepper fruits (n = 8) were wounded with a sterile needle and a 12 µl drop of conidial suspension (4 x 105/ml) of each isolate was pipetted onto the wound. Control fruit received sterile water. The fruit were incubated at 100% RH in sealed containers at 20-22°C. After 7-10 d incubation small dark sunken spots developed, and by 14 d lesions formed with velvety conidial growth similar to the ones originally observed. The fungus was successfully isolated again from the lesions onto PDA and confirmed as C. cladosporioides based on colony and conidial morphology. No symptoms developed on fruit inoculated with sterile water. The pathogenicity test was repeated two times with similar results. C. cladosporioides is a cosmopolitan pathogen and has been cited causing disease on numerous crops worldwide (Bensch et al., 2012). This species was reported causing tomato leaf spot in Mexico (Robles-Yerena et al., 2019) and black mould of tomato fruit in Australia (Ma et al., 2020). Cladosporium herbarum was found causing fruit rot of bell pepper in the U.S. (Ramsey and Heiberg, 1952). Sweet peppers are widely grown in Israel, and have high economic value as an export crop. Control measures to limit losses from C. cladosporioides postharvest may be necessary if conditions are favorable for disease during prolonged storage and refrigerated transport.

Investigation of CDH1 germline mutations in Turkish patients with Kaposi’s sarcoma

Abstract Objectives Kaposi’s sarcoma (KS) develops from the lining cells of blood or lymphatic vessels and may appear as red, purple, brown, or black lesions. E-cadherin, CDH1, is a cell adhesion molecule located on the surface of epithelial cells. CDH1 gene expression is downregulated in several cancers and is considered a tumor suppressor gene involved in epithelial-mesenchymal transition in carcinomas. Loss of CDH1 gene expression is observed in many carcinomas, mainly diffuse gastric carcinomas and lobular breast carcinomas, as well as skin tumors. This study investigates the CDH1 germline mutations in HIV-negative (Human Immunodeficiency) Virus KS patients in the Turkish population. Methods The study examined 25 peripheral blood mononuclear cells from KS patients using the Sanger sequencing technique. Results Sixteen exons of the CDH1 gene were sequenced, and a pathogenic functional germline mutation, HET c.2245C > T, p.(Arg749Trp) rs776975632, NM _004360.5, was identified in a patient with a family history of gastric and breast cancer with a high number of lesions compared to other KS patients. Discussion: KS patients with a family history of cancer could be screened for CDH1 gene and cancer-related-gen variants in the future. Conclusions KS is a rare malignancy, and genetic analysis will benefit KS patients. Further studies are needed to describe better the variations detected in a large number of KS patients in this study.

First Report of Anthracnose Disease on avocado (Persea americana) caused by Colletotrichum fructicola in China.

In September 2019, anthracnose-like symptoms were observed on fresh avocado fruits cv. "Hass", which were imported from Peru (Mission Produce, Inc.) and purchased at Ganfuyuan store, Guangzhou, China. After being stored for 5 days at room temperature, initial black specks developed into larger brown or black lesions on fruits, and salmon-colored conidial mass in the lesions were observed. To isolate and identify the pathogen, small pieces (5 mm × 5 mm) were excised from the lesion margins of the fruits, which were surface sterilized by 1% NaOCl (1 min), 70% ethanol (30 s) and then washed twice with sterile distilled water (SDW). After sterilization, the tissues were cultured on Potato Dextrose Agar (PDA) to obtain the pure strain NYG. The colonies grown on PDA for 7 days appeared to be cottony, white to pale gray with the presence of a conidial mass. Conidia 11.7-19 × 3.6-6.4 μm (n = 50), hyaline, aseptate, straight and cylindrical with rounded ends, Appressoria 6.2-11.9 × 4.5 × 7.4 μm (n = 50), brown to dark brown in different shapes. Perithecia were thick-walled and globose with a prominent, narrow neck. Asci 31.5-55 × 6-12.5 μm (n = 15), 6-8 spored, clavate to cymbiform. Ascospores 5-18 × 4.5-6 μm (n = 25), hyaline, large guttulate at the centre, slightly curved, rounded ends. Based on the morphological characteristics, the strain NYG was identified as Colletotrichum fructicola (Prihastuti et al. 2009). The identity of the strain was confirmed by means of multi-locus gene sequencing. The genomic DNA was extracted using Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech Co., Ltd., China). The internal transcribed spacer (ITS) rDNA region, actin (ACT), chitin synthase (CHS-1), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) partial genes (Templeton et al. 1999, Carbone et al. 1999) were amplified and sequenced, which were deposited in GenBank (OL413493, OL517766, OL517768, OM141126, OL517767). BLASTN analysis revealed that DNA sequences of the isolate showed 100% identity with those of C. fructicola (MT476840.1, MK208862.1, MZ965245.1, JX009665.1, MN982434.1), respectively. A phylogenetic tree analysis based on the concatenated sequences confirmed the isolate as C. fructicola. Pathogenicity was tested by infecting the fresh healthy avocado fruits with the isolated strain NYG. The fruits were surface sterilized, three unwounded and wounded avocados fruits were respectively inoculated with 10 l of conidial suspension (1×106 conidia/ml) by the drop inoculation method. Control fruits were inoculated with SDW containing Tween 20 (1 μl/ml H2O), respectively. All inoculated fruits were incubated at 25°C in the dark. Anthracnose symptoms were observed on the wounded and unwounded fruits after 3 to 5 days post inoculation, respectively. No symptoms were observed in the control on both the wounded and unwounded fruits. The pathogenicity test was performed in duplicate. The inoculated fungus was reisolated from the infected fruits and confirmed as C. fructicola, thus confirming Koch's postulates. C. fructicola represents an important fungal pathogen in several plantations worldwide (Farr et al. 2020), for example, the avocado fruits in Mexico (Dionicio, et al. 2018) and New Zealand (Hofer et al. 2021). This is the first report of anthracnose caused by C. fructicola on imported avocado fruits in China. The results of this study can not only help establish effective quarantine measures against anthracnose disease for imported avocado fruits in China, but also provide important reference to prevent the spread of this disease on China's domestic avocados.

First Report of Postharvest Fruit Rot in Solanum muricatum Aiton Caused by Alternaria alternata in southwest China.

Solanum muricatum is native to South America and well known for its sweet, attractive, nutritious fruits. S. muricatum has been cultivated in China since the 1980s and increasingly popular (Li et al. 2015). In November 2021, an unknown fruit rot was observed in Shilin County of Yunnan Province (24.77 °N, 103.28 °E). The incidence of this disease was about 16% of 500 postharvest S. muricatum fruits after 7 d in storage room (25°C, 90% relative humidity). The initial symptoms were small brown spots on the fruit surface, which gradually expanded into irregular brown or black lesions, and gray-white mold developed in the center of the lesions, eventually the fruit turned rot. To isolate the pathogen, ten fruits with typical symptoms were collected and surface-sterilized with 75% ethanol for 45 s. Small fragments (5 × 5 mm) from the margin of lesions on fruit were disinfected with 1% sodium hypochlorite for 60 s, washed three times with sterile water then transferred to potato dextrose agar (PDA), and incubated at 28 ± 1℃ for 3 days (Li et al. 2022). Two fungal isolates with the same morphology were obtained and purified by single-spore isolation method. The colony was covered with thick fluffy aerial mycelia and the center was dark brown or black with white margins. Conidia were brown, pyriform or ellipsoid, with 1 to 3 longitudinal and 2 to 6 transverse septa, 15.12 to 34.01 × 6.90 to 12.73 μm (21.22 × 9.69 μm on average, n=50) in size. These morphological characteristics were consistent with Alternaria alternata (Li et al. 2015; Xiang et al. 2021; Alberto. 1992). For molecular identification, genomic DNA was extracted from a representative isolate, and primers ITS1/ITS4 (Gardes et al. 1993), TEF-F/TEF-R (Lawrence et al. 2013), Alt-F/Alt-R (Hong et al. 2005), GPD-F/GPD-R (Berbee et al. 1999) and EPG-F/EPG-R (Peever et al. 2004) were used to amplify the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF), Alternaria major allergen (Alt a1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and endo-polygalacturonase (endoPG), respectively. The obtained DNA sequences (ITS: OM049821; TEF: OM069656; Alt a1: OM069655; GAPDH: OM069654 and endoPG: OM069653) showed over 99% homology with that of A. alternata (GenBank Accession No. MN856355.1 (565/573 bp); MN258023.1 (267/267 bp); KY923227.1 (491/501 bp); LC131645.1 (608/609 bp) and MN698284.1 (452/454 bp)). A phylogenetic tree based on the combined ITS, TEF, Alt a1, GAPDH, and endoPG sequences using the maximum likelihood methods with Kimura 2-parameter model, bootstrap nodal support for 1000 replicates in MEGA7.0 (Li et al. 2019) revealed that the isolate was assigned to A. alternata. To confirm pathogenicity, 10 μL spore suspension (1.0 × 106 conidia/ml) obtained from 7-day-old PDA cultures of each isolate were inoculated on 15 needle-wounded and 15 non-wounded surface-disinfected fruits, respectively. Healthy fruits were inoculated with sterile water as controls and the experiment was repeated 3 times. All fruit were incubated at 25 ± 1℃, 90% relative humidity. After 7 days, all the wounded and non-wounded fruit inoculated with A. alternata showed similar symptoms to those observed on the previously fruits, while the control fruits remained healthy. The same pathogen was again isolated from the inoculated fruits, thus Koch's postulates were fulfilled. A. alternata causing fruit rot of Prunus avium and Mangifera indica in China were reported in previous studies (Ahmad et al. 2020; Liu et al. 2019). As far as we know, this is the first report of postharvest fruit rot on S. muricatum caused by A. alternata in southwest China. This work provides a basis for the development of control strategies of the disease in the future.