Phyllosticta Capitalensis Research Articles

Identification and fungicide screening of Phyllosticta capitalensis causing leaf spot on sweet viburnum in China

Sweet viburnum (Viburnum odoratissimum Ker-Gawl.) is a widely used ornamental plant, which has dense branches and leaves, and fast spreading and evergreen habit. In October 2022, leaf spot symptoms were observed in a hedge of sweet viburnum in Yuanshi Garden, Ningbo, China. Fungi were isolated from symptomatic leaves, and were identified using morphological characteristics and phylogenetic analyses of partial sequences of internal transcribed spacer (ITS), actin (act), and translation elongation factor 1-alpha (tef1-α), and were evaluated in pathogenicity tests. The causal agent of sweet viburnum leaf spot was identified to be Phyllosticta capitalensis. Effects of seven fungicides on P. capitalensis were assessed in vitro. Fungicide EC50s (mg L-1) against P. capitalensis were: 270.77 for 75% chlorothalonil (WP); 0.02 for 250 g L-1 azoxystrobin SC; 0.27 for 10% difenconazole WDG; 0.02 for 75% trifloxystrobin + tebuconazole WDG, 9.03 for 35% fluopyram + tebuconazole SC, 5.90 for 500 g L-1 fluazinam SC, and 89.11 for 10% prothioconazole SC. Among these, azoxystrobin SC and trifloxystrobin + tebuconazole WDG could be used for control of viburnum leaf spot. This is the first report of P. capitalensis causing leaf spot of sweet viburnum, and this study provides guidance for chemical management sweet viburnum leaf spot, and on other host plants.

First Report of Phyllosticta capitalensis Causing Postharvest Brown Spot Disease on Guava Fruit in Thailand.

Guava (Psidium guajava L.) is a popular fruit crop that is widely cultivated in Thailand. In November 2023, brown spot disease on guava was observed during postharvest storage at 22 to 31°C and 70 to 75% relative humidity over a period of 3 to 7 days in Fang District, Chiang Mai Province, Thailand. The disease incidence was ~20% of 100 fruits per pallet box. The disease severity on each fruit ranged from 40 to 70% of the surface area affected by lesions. The symptoms appeared as circular to irregular brown to dark brown spots, ranging from 5 to 30 mm in diameter. Fungi were isolated from lesions using a single conidial isolation method (Choi et al. 1999). Two fungal isolates (SDBR-CMU497 and SDBR-CMU498) with similar morphology were obtained. Colonies on potato dextrose agar (PDA) and malt extract agar (MEA) were 65 to 67 and 29 to 38 mm in diameter, respectively after incubation for 1 week at 25°C. Colonies on PDA and MEA were flat, slightly undulate, greenish gray in the center, greyish green at the margin; reverse black. Both isolates produced asexual structures. Pycnidia were black, granular, and grouped. Conidiogenous cells were hyaline, subcylindrical to cylindrical, 8.5 to 17.5 × 3 to 5.5 µm. Conidia were single-celled, hyaline, obovoid to ellipsoid, 5.2 to 9.4 × 3.6 to 7.5 µm (n = 50), smooth-walled, with a single apical appendage. Morphologically, both isolates resembled Phyllosticta capitalensis (Wikee et al. 2013). The internal transcribed spacer (ITS) region, large subunit (nrLSU), translation elongation factor 1-alpha (tef1-α), actin (act), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified using primer pairs ITS5/ITS4, LROR/LRO5, EF1-728F/EF2, ACT-512F/ACT-783R, and GPD1-LM/GPD2-LM, respectively (White et al. 1990; Zhang et al. 2022). Sequences were deposited in GenBank (ITS: PP946770, PP946771; nrLSU: PP948677, PP948678; tef1-α: PP948012, PP948013; act: PP948014, PP948015; GAPDH: PP948016, PP948017). Maximum likelihood phylogenetic analyses of the concatenated five genes identified both isolates as P. capitalensis. Thus, both morphology and molecular data confirmed the fungus as P. capitalensis. To confirm pathogenicity, healthy commercial guava fruits cultivar Kim Ju were surface disinfected by 0.1% NaClO for 3 min, rinsed three times with sterile distilled water, and wounded (Cruz-Lagunas et al. 2023). Conidia were collected from 2-week-old cultures on PDA and suspended in sterile distilled water. Fifteen microliters of a 1 × 106 conidia/ml suspension were dropped onto the wounded fruits. Mock inoculations were used as a control with sterile distilled water. Ten replications were conducted for each treatment and repeated twice. The inoculated fruits were stored in individual sterile plastic boxes at 25°C with 80 to 90% relative humidity. After 7 days, all inoculated fruits exhibited brown to dark brown lesions, while control fruits were asymptomatic. Phyllosticta capitalensis was consistently reisolated from the inoculated tissues on PDA to complete Koch's postulates. Prior to this study, P. capitalensis was known to cause brown or black spot disease on guava fruits cultivated in fields in China (Liao et al. 2020), Egypt (Arafat 2018), and Mexico (Cruz-Lagunas et al. 2023). To our knowledge, this is the first report of P. capitalensis causing postharvest brown spot disease on guava fruit in Thailand. The results will inform epidemiological investigations and future approaches to managing this disease.

Morphological and multi-gene phylogenetic analyses of two new species of Phyllosticta (Phyllostictaceae, Botryosphaeriales) from Southwestern China

Phyllosticta is an important genus of pathogenic fungi with worldwide distribution. Phyllosticta species also occur as endophytes and saprobes on various plant hosts. Multi-gene phylogenetic analysis based on sequence data of ITS, LSU, TEF1 and morphological evidence supported the introduction of two new species, which were isolated from dead leaves in China. Phylogenetically, the new species, P. savannaensis and P. ovalina, formed two well-separated lineages in the Phyllosticta capitalensis and Phyllosticta cruenta species complexes, respectively. P. savannaensis is closely related to P. capitalensis, but formed a separate branch (BS= 100%/BYPP= 1.0), while P. ovalina and P. hubeiensis clustered together with high support (BS= 100%/BYPP= 1.0). Both new species exhibited the typical characteristics of the genus Phyllosticta but could be distinguished from closely related species. P. savannaensis can be differentiated from the sexual stage of other Phyllosticta species by the length to width ratio of the ascospores and shape of appendages. The ascospores of P. ovalina are oval, which distinguish it from other species of Phyllosticta.

Endophytic fungi-assisted biomass synthesis of eco-friendly formulated silver nanoparticles for enhanced antibacterial, antioxidant, and antidiabetic activities

Fungi-mediated biosynthesis of silver nanoparticles is one of the promising nanomanufacturing processes. Using endophytic fungi isolated from potential medicinal plants has emerged as a novel approach for producing several metal oxide nanoparticles in a green and cost-effective manner. The present study validates the biomimetic synthesis of silver nanoparticles of endophytic fungus (Phyllosticta capitalensis Henn.) (Phyllo-AgNPs) isolated from the leaves of Annona muricata and the effect of process variables, i.e., reductant concentration, ratio of AgNO3 to reductant concentration, reaction time and reaction pH to achieve maximum efficiency using the factorial design approach. The nanoparticle formation was confirmed by absorption peaks between 400 nm and 500 nm, followed by their characterization by UV–visible spectroscopy, TEM, SEM-EDX, XRD, particle size, and zeta potential analysis. The in-vitro antioxidant, antidiabetic and antimicrobial potential (agar well dfiffusion and minimum inhibitory concentration) of Phyllo-AgNPs at various concentrations were assessed. The Phyllo-AgNPs scavenged the 2,2-diphenyl-1-picrylhydrazyl radical and inhibited the activity of α-amylase and α-glucosidase enzymes at an IC50 of 31.75 ± 0.70, 39.32 ± 1.12 and 28.56 ± 1.08 μg/mL, respectively. The antimicrobial potential was determined against Listeria monocytogenes, Staphylococcus aureus, Escherichia coli and, Salmonella typhimurium. The study revealed the therapeutic potential of Phyllo-AgNPs against pathogenic microorganisms. Additionally, the in-vitro antioxidant and antidiabetic activities showed improved results compared to the standard (ascorbic acid and acarbose). By taking note of this, Phyllo-AgNPs can be further studied for pre-clinical trials, which would be beneficial in developing novel medicines with no adverse effects for the benefit of mankind.

Molecular characterization and evaluation of phytochemical constituents of endophytic fungi derived from Mitracarpus scaber Zucc. (Rubiaceae)

In quest to ameliorate the issues of recurring incidences of multidrug-resistant organisms and over-exploration of plants, researchers have delved into researches involving endosymbiotic microorganisms, known as endophytes. These are microorganisms inhabiting the internal part of plants, which have been verified to possess great potentials of bioengineering novel products for therapeutic purposes. The aim of this study was to isolate and identify endophytic fungal species present in Mitracarpus scaber, molecularly characterize the pure isolates, and test for phytochemical compounds present. Freshly collected non-diseased leaves of M. scaber were subjected to a four-step surface sterilization. Thereafter, they were sliced into fragments of 1 cm-length, exposing the leaf blades and the midribs, and were aseptically inoculated on sterilized malt extract agar, containing chloramphenicol (500 mg/l), in Petri dishes. Hyphal tips of actively growing fungi from the plant material were harvested and further sub-cultured for purification and isolation. Segments were aseptically cut from the actively growing pure isolates, on malt extract agar and inoculated onto the fermentation medium (sterilized local rice), in 500 ml Erlenmeyer flasks, which were properly sealed with sterile cotton and kept at static condition at 25 oC for 21 days. Ethyl acetate was used to extract the metabolites from the end products of the fermentation processes. Four isolates obtained from the endophytic fungi (EDF), present in the leaf of M. scaber, were subjected to molecular identification. The DNA extraction was done using Zr fungal/bacterial DNA miniprep. The extracted DNA was amplified through PCR, and sequenced. The resultant sequences were compared with GenBank database, using Basic Local Alignment Search Tool. The result of phytochemical screening of the extracts revealed the presence of flavonoids, tannins and phenols, in large amounts; terpenoids, alkaloids and cardiac glycosides, in moderate amounts; steroids, hydrogen cyanide and saponins in very low quantities, while that of molecular characterization identified four organisms: Penicillium sclerotiorum, Clasporium cladosporiodies, Cryptococcus nemorosus, and Phyllosticta capitalensis.

Diversity of endophytic fungi from medicinal plant Oxalis latifolia and their antimicrobial potential against selected human pathogens

Endophytic fungi that inhabit medicinal plants are microbial resources renowned for having compounds analogous to those produced by their host plants. This study aimed to describe the diversity of endophytic fungi found in Oxalis latifolia Kunth. To better understand the diversity of foliar endophytic fungi found in the leaves of the medicinal plant Oxalis latifolia, we isolated and characterized endophytic by using both morphological and molecular methods employing ITS markers. The antimicrobial activity of endophytic fungi against common human pathogens Escherichia coli, Staphylococcus aureus, and Bacillus subtilis was also investigated. A Total of 16 endophytic fungi were successfully isolated from leaves and classified into five orders of Pezizomycotina based on the phylogenic analyses; Xylariales (56%), Diaporthales (19%) Sordariales (6%), Glomerellales (13%) and Botryosphaeriales (6%). The antimicrobial activity of crude extracts from fungal endophyte against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis revealed that three isolates; N. aurantiaca, Phyllosticta capitalensis N. oryzae were the most potent, while Colletotrichum karstii and N. sphaerica displayed no growth inhibition property against the tested organism. The diversity indices were calculated by using the Shannon-Wiener, Margalef, and Simpson indices. The diversity indices analysis revealed an abundance of species diversity, where the dominant species were Nigrospora oryzae, N. sphaerica, and Colletotrichum karstii. This study describes the diversity of endophytic fungi found in O. latifolia and emphasizes their potential as a source of novel bioactive compounds. More research on phytochemical composition and antimicrobial activity is ongoing to correlate the traditional uses and scientific findings.

First report of Phyllosticta capitalensis causing leaf brown spot of Ligustrum japonicum in China

First report of Phyllosticta capitalensis causing leaf brown spot of Ligustrum japonicum in China

Meroterpenoids with divers' rings systems from Phyllosticta capitalensis and their anti-inflammatory activity

Four undescribed sesquiterpene-shikimates (1–4), eight undescribed monoterpene-shikimates (5–12), together with two known ones were isolated and identified from the 95% ethanol extract of the plant endophytic fungus Phyllosticta capitalensis cultured in rice medium. Capitalensis A (1) was identified as the first sesquiterpene-shikimate-conjugated spirocyclic meroterpenoid degradation product, while capitalensis B (2) is a sesquiterpene-shikimate-conjugated spirocyclic meroterpenoid with a unique D-ring formed by a C-2–O–C-9′ connection. The structures of these previously undescribed compounds were elucidated by multiple techniques, including IR, HR-ESI-MS, and NMR analysis. Furthermore, their absolute configurations were established through the comprehensive approach that involved the calculations of ECD spectra, optical rotation values, and single-crystal X-ray analysis. Moreover, the anti-inflammatory activity of all isolated compounds was evaluated using a lipopolysaccharide (LPS)-induced inflammation model in BV2 microglial cells. Meanwhile, these compounds exhibited activity in inhibiting NO production. Four compounds, capitalensis C (3), capitalensis D (4), 15-hydroxyl tricycloalternarene 5b (13) and guignarenone A (14) showed strong inhibitory effects with IC50 values of 21.6 ± 1.33, 12.2 ± 1.08, 18.6 ± 1.27, and 15.8 ± 1.20 μM, respectively. In addition, the structure-activity relationship of the anti-inflammatory activity of the compounds was discussed.

Phylloremediation of pyrene and anthracene by endophytic fungi inhabiting tea leaves (Camellia sinensis (L.) Kuntze) in Sri Lanka

ABSTRACT Polyaromatic hydrocarbons (PAHs) released from vehicular emissions and oil refineries deposit on the phyllosphere, compromising the quality of leaf-based food products by posing many health issues. Nevertheless, the tea phyllosphere harbours a variety of endophytes that are highly effective at degrading polyaromatic hydrocarbons, anthracene, and pyrene. The present study attempts to analyse the pyrene and anthracene degrading capability of phyllosphere endophytic fungi that inhabit Camellia sinensis (L.) Kuntze leaves. The frequency of occurrence of endophytic fungi in different leaf tissue layers was examined using light and scanning electron microscopy (SEM). The best pyrene and anthracene degrading strains were selected based on the High-Performance Liquid Chromatography (HPLC) results, and further kinetic assays. Light microscopy and SEM observations highlighted a heterogeneous endophytic fungal distribution among leaf tissue layers; the upper epidermis had the highest fungal distribution compared to other leaf layers. HPLC results revealed that Phyllosticta capitalensis, Colletotrichum gloeosporioides, Colletotrichum siamense, Pseudopestalotiopsis chinensis, and Daldinia eschscholtzii, have higher pyrene and anthracene degradation respectively and their PAH degradation kinetics follow the first-order kinetic model. The best anthracene and pyrene degrader, P. capitalensis showed the lowest half-life. The present investigation highlights the potential of P. capitalensis, the best pyrene and anthracene degrader that can remediate PAHs deposited on the phyllosphere of tea leaves.

First report of black patch disease caused by Phyllosticta capitalensis on Litsea cubeba in China.

Litsea cubeba, an economical important tree species originally from China, produces fruit from which essential oils are extracted and extensively used in the chemical industry (Zhang et al. 2020). In August 2021, a large-scale outbreak of black patch disease was first observed on the leaves of Litsea cubeba in Huaihua (27°33'N; 109°57'E), Hunan province, China (disease incidence 78%). A second outbreak in 2022, in the same area, lasted from June to August. Symptoms consisted of irregular lesions that initially appeared as small black patches near the lateral veins. These lesions grew along the lateral veins and formed feathery patches until almost the entire lateral veins of the leaves were infected by the pathogen. The infected plants grew poorly and eventually the leaves desiccated and the tree defoliated. To identify the causal agent, the pathogen was isolated from nine symptomatic leaves from three trees. Symptomatic leaves were washed with distilled water three times. Leaves were cut into small pieces (11 cm), surface sterilized with 75% ethanol for 10s and 0.1% HgCl2 for 3 min, and then washed 3 times in sterile distilled water. Surface disinfected leaf pieces were placed onto potato dextrose agar (PDA) medium with cephalothin (0.2 mg/ml) and incubated at 28°C for 4-8 days (about 16h light, 8h dark). Seven morphologically identical isolates were obtained, from which five were selected for further morphological examination and three for molecular identification and pathogenicity test. Strains from grayish white colonies with a granular surface and grayish black wavy edges; bottom of the colonies turned black over time. Conidia were hyaline and nearly elliptical, unicellular. The sizes of conidia ranged from 8.59 to 15.06 μm (n=50) in length and 3.57 to 6.36 μm (n=50) in width. These morphological characteristics are consistent with the description of Phyllosticta capitalensis (Guarnaccia et al. 2017, Wikee et al. 2013). To further confirm the identity of this pathogen, genomic DNA of three isolates (phy1, phy2 and phy3) were extracted to amplify the internal transcribed spacer (ITS) region, the 18S rDNA region, the transcription elongation factor (TEF), and actin (ACT) gene with ITS1/ITS4 (Cheng et al. 2019), NS1/NS8 (Zhan et al. 2014), EF1-728F/EF1-986R (Druzhinina et al. 2005) and ACT-512F/ACT-783R (Wikee et al. 2013) primers, respectively. Sequence similarity indicated that these isolates were highly homologous to Phyllosticta capitalensis. The ITS (Genbank No. OP863032, ON714650 and OP863033), 18S rDNA (Genbank No. OP863038, ON778575 and OP863039), TEF (Genbank No. OP905580, OP905581 and OP905582) and ACT (Genbank No. OP897308, OP897309 and OP897310) sequences of isolates Phy1, Phy2 and Phy3 shared up to 99%, 99%, 100% and 100% similarities with their counterparts (Genbank No. OP163688, MH051003, ON246258 and KY855652) in Phyllosticta capitalensis, respectively. To further confirm their identity, a neighbor-joining phylogenetic tree was generated using MEGA7. Based on morphological characteristics and sequence analysis, the three strains were identified as P. capitalensis. To fulfill Koch's postulates, conidial suspension (1×105 conidia per mL) collected from three isolates were independently inoculated on artificially wounded detached leaves and leaves on trees of Litsea cubeba. Leaves were inoculated with sterile distilled water as negative controls. The experiment was repeated three times. All pathogen-inoculated wounds exhibited necrotic lesions within 5 days on detached leaves and 10 days on the leaves growing on trees after inoculation, whereas no symptoms were observed on the controls. The pathogen was exclusively re-isolated from the infected leaves and showed identical morphological characteristics to those of the original pathogens. P. capitalensis is a destructive plant pathogen that has been shown to cause leaf spots or black patch symptoms on variety of host plants around the world (Wikee et al. 2013), including oil palm (Elaeis guineensis Jacq.), tea plant (Camellia sinensis), Rubus chingii and castor (Ricinus communis L.). To our knowledge, this is the first report of black patch disease of Litsea cubeba caused by P. capitalensis in China. This disease causes severe leaf abscission in fruit development stage of Litsea cubeba and leads to a large amount of fruit drop.

First Report of Leaf Blotch Caused by Phyllosticta capitalensis on Daphne odora in China.

Daphne odora Thunb. an evergreen shrub with scented flowers, is used for ornamental purposes but it also has medicinal benefits (Otsuki, et al. 2020). In August 2021, leaf blotch symptoms were observed on roughly 20% of leaves of D. odora var. marginata plants in Fenghuangzhou Citizen Park, Nanchang city (28°41'48.12″ N, 115°52'40.47″ E), Jiangxi Province, China. Brown lesions first appeared on the edges of leaves, which eventually dried and died (Fig. 1A). For fungal isolation, 12 symptomatic leaves were randomly collected, the edges between diseased area and healthy area were cut into small pieces (4×4 mm), surface-sterilized by dipping in 70% ethanol for 10 s and 1% sodium hypochlorite for 30 s, and then rinsed three times with sterile distilled water. Leaf pieces were then plated on potato dextrose agar (PDA) and incubated at 28 ℃ for 3-4 days. A total of 10 isolates were recovered from the diseased leaves. The pure colonies of all fungal isolates had similar characteristics, and three isolates were randomly selected (JFRL 03-249, JFRL 03-250 and JFRL 03-251) for further study. Colonies of this fungus were gray and uneven, with a granular surface, and irregular white edges, finally turning black on PDA (Fig. 1B, C). Pycnidia were black, globose and 54-222 μm in diameter (Fig. 1D). Conidia were hyaline, single-celled, and nearly elliptical, which ranged from 7 to 13 × 5 to 7 μm (n=40) (Fig. 1E). These morphological characteristics were consistent with those described for the fungus Phyllosticta spp. (Wikee et al. 2013a). To confirm the fungal identity, the internal transcribed spacer (ITS) region, actin (ACT), translation elongation factor 1-alpha (TEF1-a), glyceradehyde-3-phosphate dehydrogenase (GPD) and RNA polymerase II second largest subunit (RPB2) genes were amplified using primers ITS5/ITS4, ACT-512F/ACT-783R, EF-728F/EF2, Gpd1-LM/Gpd2-LM and RPB2-5F2 /fRPB2-7cR, respectively (Wikee et al. 2013b). The sequences of the selected isolates were 100% identical. Hence, sequences of one representative isolate JFRL 03-250 were deposited in GenBank (OP854673, ITS; OP867004, ACT; OP867007, TEF1-a; OP867010, GPD; and OQ559562, RPB2). BLAST search analysis in GenBank showed 100% similarity with those of P. capitalensis (GenBank accession nos. ITS, MH183391; ACT, KY855662; TEF1-a, KM816635; GPD, OM640050 and RPB2, KY855820). From a phylogenetic perspective, a maximum likelihood phylogenetic tree was constructed by using IQtree V1.5.6 based on multiple sequences (ITS, ACT, TEF1-a, GPD and RPB2) (Nguyen et al. 2015), and the cluster analysis resulted the representative isolate JFRL 03-250 within a clade comprising Phyllosticta capitalensis (Fig. 2). Based on morphological and molecular characters, the isolate was identified as P. capitalensis. To confirm pathogenicity and fulfill Koch's postulates, 6 healthy potted plants were inoculated with 1× 106 conidia/ml suspension of isolate JFRL 03-250 by spraying on the leaves, whereas 6 plants were sprayed with sterile distilled water to serve as control. All potted plants were incubated at 28°C, 80% relative humidity and 12-h light/12-h dark alternating conditions in a climate cabinet. After 15 days, similar symptoms were observed in the inoculated leaves as in the field (Fig. 1F), whereas control leaves remained asymptomatic (Fig. 1G) and P. capitalensis was successfully re-isolated from the symptomatic leaves. Previously, P. capitalensis has been reported to cause brown leaf spot disease of various host plants around the world (Wikee et al. 2013b). However, to our knowledge, this is the first report of brown leaf spot caused by P. capitalensis on D. odora in China.

First report of Phyllosticta capitalensis causing brown spot disease on guava fruits (Psidium guajava) in Mexico.

The guava (Psidium guajava L.) is a plant native to the tropical region of America. In Mexico, the area established with guava cultivation is 20,525 ha (SIAP 2021). Guava is commonly consumed as fresh fruit, being rich in nutrients such as vitamins and minerals (Murthy et al. 2020). During October 2020, in the municipality of Cocula (18.207835N, 99.670322W, 595 m above sea level), Guerrero, Mexico, severely infected immature guava fruits were observed. The incidence of disease in 150 sampled fruits was 12%. Were collected fifteen symptomatic fruits. The symptoms were circular to irregular dark brown spots that varied in size (0.5 to 2.5 cm). From symptomatic fruits, tissues were cut approximately 3 x 3 mm and disinfested with 1% NaOCl, washed three times with sterile distilled water, and transferred to PDA medium amended with streptomycin and tetracycline, and incubated at 28°C. Developing colonies were retransferred to new culture PDA medium, and purified by hyphal tip technique. Two representative isolates (PHYGUA7 and PHYGUA3) were selected for morphological and molecular characterization. After 15 days in PDA at 28°C in an incubator, colonies were flat, irregular, granular and greenish gray, pycnidia were black, granular, and grouped. The conidia were hyaline and ellipsoid, unicellular and smooth-walled, 7-11×5-6.5 μm (n=50), these characteristics were consistent with those described for the fungus Phyllosticta capitalensis (Wikee et al. 2013). Molecular identification was performed by partially sequencing the internal transcribed spacer gene (ITS), the actin gene (ACT), and the translation elongation factor 1-alpha (EF-1α) gene, using primers ITS1 and ITS4, ACT-512F/ ACT-783R, and EF1-728F/EF1-986R, respectively (White et al. 1990; Carbone and Kohn 1999). The resulting sequences were deposited in GenBank (PHYGUA7: OP810947, PHYGUA3: OP810948 for ITS, PHYGUA7: OP819845, PHYGUA3: OP819846 for ACT, and PHYGUA7: OP819847, PHYGUA3: OP819848 for EF-1α). Phylogenetic analysis using maximum likelihood concatenated sequences ITS, ACT and EF-1α with MEGA X, indicated that PHYGUA7 and PHYGUA3 isolated grouped with P. capitalensis (CPC 18848 type strain). For pathogenicity test of P. capitalensis, 15 healthy immature fruits in a field experiment in the fruits on the trees, and 15 healthy mature guava fruits (detached fruits) were superficially disinfected with 70% ethanol, wounded with a sterile toothpick, and inoculated at two equidistant points by inserting PHYGUA7 isolate mycelium. As a control treatment, 10 healthy immature fruits and 10 healthy mature fruits were only injured with a sterile toothpick. After 3 days symptoms were observed in mature fruits and numerous dark pycnidia developed, and seven days later symptoms were observed in immature fruits in all the points inoculated with the PHYGUA7 isolate, similar to the symptoms observed in the field. The control fruits remained asymptomatic. The fungus P. capitalensis was re-isolated from inoculated fruits, thus confirming Koch's postulates. In Mexico P. capitalensis has been reported in Mangifera indica, Epidendrum sp., and Schomburgkia tibicinis (Farr and Rossman, 2022). In Egypt and China P. capitalensis causes black spot on guava fruits (Arafat 2016; Liao et al. 2020). To our knowledge, this is the first report of P. capitalensis as the cause of brown spot on immature guava fruit in Mexico. This research provides relevant information to the design of disease management strategies.

First report of Phyllosticta capitalensis causing leaf spot of Mahonia fortunei in China.

Mahonia fortunei, belonging to the Berberidaceae family, is widely cultivated in fields, parks, courtyards, and roadsides for its excellent ornamental characteristics and medicinal values in southern China (Yu and Chung 2017). In May 2021, leaf spots were observed on nearly 60~80% of M. fortunei plants growing in Chongqing Normal University campus (29°36'42″N; 106°17'59″E) from Chongqing City, China. The typical symptoms on leaves were irregular spots with gray centers, brown edges, and chlorotic halos, about 1 to 7 mm in diameter, and eventually coalesced forming larger necrotic areas. Twenty symptomatic leaves were randomly sampled from five diseased plants. Tissues were cut from the lesion margins and surface sterilized in 75% ethanol for 1 min, rinsed thrice with sterile water, dried on sterilized paper, plated on potato dextrose agar (PDA) plates, and incubated at 25°C for 7 days in the dark. A total of 20 isolates were obtained from the infected leaves. Pure colonies of all fungal isolates had similar characteristics, and three isolates were randomly selected (SD11, SD18, SD19) for further study. Colonies of this fungus were olivaceous greenish to olivaceous black with a granular surface, and irregular light olive edges, finally turning black on PDA. Pycnidia were black, globose, granular, and in clusters. Conidia (n=30) were hyaline, aseptate, unicellular, obovoid to ellipsoid, narrow end with single apical appendage, and 7.5~11.2 × 4.5 ~6.5 μm. The DNA of three isolates were extracted and the internal transcribed spacer (ITS) region, actin (ACT), and translation elongation factor 1-α (TEF1) genes were amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990), ACT512F/ACT783R, and ER728F/EF986R (Carbone and Kohn 1999), respectively. The sequences of three isolates were 100% identical, and one representative isolate SD18 were deposited in GenBank (ON231754, ITS; ON246259, ACT; and ON246258, TEF1). Sequence analysis revealed that the consensus sequences of ITS, ACT, and TEF1 of isolate SD18 was 99 to 100% identical to each sequence of an Indonesian strain (CBS 117118) of P. capitalensis from Musa acuminate (FJ538339 for ITS, FJ538455 for ACT, FJ538397 for TEF1). Phylogenetic analysis using Maximum Likelihood and concatenated sequences (ITS+ACT+TEF1) with MEGA7 placed isolate SD18 in P. capitalensis with 100% bootstrap support. Based on these morphological and molecular characteristics, the isolates were identified as P. capitalensis (Wikee et al. 2013). To fulfill Koch's postulates, 8 healthy potted plants were inoculated with 106 conidia/ml suspension of isolate SD18 by spraying the leaves, and another 8 plants were sprayed with sterile distilled water as control. All plants were covered with plastic bags for two days and then arranged in a greenhouse with 80% relative humidity at 25°C. The pathogenicity test was repeated thrice. After 18 days inoculation, the similar symptoms were observed on the inoculated plants, whereas control plants remained healthy. The pathogen was reisolated from symptomatic tissue and identified as P. capitalensis by the methods described above. P. capitalensis has been reported causing leaf spot on various host plants around the world (Wikee et al. 2013), recently found on tea plant, castor, and oil palm (Cheng et al. 2019; Tang et al. 2020; Nasehi et al. 2020). This is the first report of P. capitalensis causing leaf spot on M. fortune in China, and will establish a foundation for controlling the disease.

First Report of Leaf Spot Caused by Phyllosticta capitalensis on Alpinia oxyphylla Miq. in China.

Alpinia oxyphylla Miq. is mainly distributed in Hainan, Guangdong and Guangxi provinces of China. Between July and August 2021, a leaf spot disease was observed in Ledong, Hainan Province, China (18°70'20.50″ N, 109°25'25.47″E) on A.oxyphylla. The incidence of infected leaves ranged from 8% to 10%, and the incidence rate of infected plants was about 50%. Symptoms appeared as primary yellow-brown withered spots on the diseased leaves, which further developed into irregular red-brown spots. The center of the lesions was gray-black, and the tissue was irregularly necrotic, ruptured or perforated, and there were yellow chlorotic halos around the edges of the lesions (Figure 1A). Tissues 5 mm in diameter were taken from the junction of diseased and healthy tissue for pathogen isolation, Successively, a total of 8 isolates were obtained from the affected leaves. Three single spore isolates (YZ-HN-001, YZ-HN-043 and YZ-HN-051) were obtained and confirmed to be identical based on morphological characteristics. Therefore, the representative isolate YZ-HN-001 was selected for morphological and molecular identification. On Potato Dextrose Agar(PDA), the colony was gray-white at first and gradually turned dark green to dark brown with lead gray on the back, growth was slow, and mycelium was short and dense (Figure 1B and Figure 1C). Pycnidia were epiphyllous, globose, brown (about 120-140 µm in diameter), and conidia were elliptical, colorless, single celled and smooth (8-12×4-7 µm) (Figure 1D). Molecular identification was performed by partially sequencing the internal transcribed spacer gene (ITS), 18S rRNA gene and the actin gene (ACT) by using the primers ITS1/ITS4 (White et al. 1990), EF4/Fungi5 (Khodaparase et al. 2005) and ACT-512F/ACT-783R (Carbone and Kohn. 1999). The sequences of the amplified fragments were deposited in GenBank, the ITS sequence (ON005130, 616 bp) showed 100% identity with Phyllosticta capitalensis strain CGMCC3.14345 (JN791605.1), the 18S rRNA sequence (ON005129, 541 bp) showed 99% identity with P. capitalensis isolate MUCC0029 (AB454185.1) and the ACT sequence (ON049348, 251 bp) showed 100% identity with P. capitalensis strain DZSN202005-2 (MW533248.1). A phylogenetic analysis was conducted in MEGA X using the neighbor-joining method and showed that isolate YZ-HN-001 clustered together with P. capitalensis (Figure 2). Based on the above morphological and molecular characteristics, the isolate was determined to be P. capitalensis. Pathogenicity tests were conducted in three replicates by inoculating surface-sterilized leaves of A. oxyphylla. The leaves were wounded and inoculated with colonized PDA plugs (5×5 mm) from 15-day-old cultures. Control leaves wounded in the same way and were inoculated with sterile PDA plugs (5×5 mm). Leaves were moisturized by spraying with sterile water every three days. After 20 days at room temperature (23 to 28℃), similar symptoms were observed in the inoculated leaves as in the field (Figure 1E), but no symptoms were observed on the control leaves (Figure 1F). The same P. capitalensis was reisolated in the inoculated leaves, confirming Koch's postulates. Phyllosticta capitalensis has been reported to cause leaf spots or black spots on various host plants around the world (Wikee et al. 2013), including on oil palm (Nasehi et al. 2020), tea plant (Cheng et al. 2019 ), and castor (Tang et al. 2020). Nevertheless, to our knowledge, this is the first report of leaf spot caused by P. capitalensis on A. oxyphylla worldwide.

First Report of Leaf Spot Caused by Phyllosticta capitalensis on Illiciumdifengpi in China.

Illicium difengpi B. N. Changet al., a shrub with aromatic odor in the Illicium genus, is extensively used as a medicinal plant in China. In June of 2020, a leaf spot on I. difengpi with incidence of about sixty percent was observed in a field located in Guilin (25°4'40"N; 110°18'21"E), Guangxi Province, China. Initialleafsymptoms were roundspotswith gray centers, surrounded by yellow halos. The spots gradually spread and merged. Six samples of symptomaticleaveswere collected from six diseased plants, and they were surface disinfested before isolation. Potato dextrose agar (PDA) was used to culture pathogens. Successively, pure cultures were obtained by transferring hyphal tips to new PDA plates. A total of 10 isolates were obtained from the affectedleaves.Two single-spore isolates (GX-1 and GX-2) were obtained and confirmed to be identical based on morphological characteristics. The representative isolate GX-2 was selected for further study on morphological and molecular characteristics. The colony of isolate GX-2 was about 4 cm in diameter on a PDA plate in 5 days, dark green with a granular surface, and irregular white edge. Conidia were hyaline, unicellular, oval, narrow at the end with a single apical appendage, and 8.2 to 13.8 × 3.7 to 7.2 µm (n= 50). Spermatia were hyaline, bacilliform with swollen ends, 3.8 to 8.9 × 1.3 to 1.9 µm (n= 50). Morphological characteristics of isolate GX-2 were consistent with the description of Phyllostictacapitalensis (Wikee et al. 2013). The internal transcribed spacer (ITS) region, translation elongation factor 1-α (tef1-α), glyceraldehyde-3-phosphate dehydrogenase (GPDH) and actin (ACT) were amplified using primers ITS1/ITS4, EF-728F/EF-986R, Gpd1-LM/Gpd2-LM and ACT-512F/ACT-783R, respectively (Wikee et al. 2013). Sequences were deposited in GenBank with accession numbers OL505439 forITS, OL539429 forACT, OL539430 fortef1-α and OL539431 for GPDH. BLAST analysis in GenBank showed that these sequences were 99 to 100% similar to the correspondingITS(MT649668),ACT(MN958710), tef1-α(MN958711) andGPDH (KU716077) sequences ofP. capitalensis. Also, the phylogenetic tree based on genes of ITS, tef1-α, GPDH and ACTby the maximum likelihood method showed that isolate GX-2 clustered together withP. capitalensis.The pathogenicity tests were carried out on a healthy 3 year-old plant in the greenhouse with 80% relative humidity at 25 °C. Four sterilized leaves were wounded with a needle and inoculated with 20 μL spore suspension (1 × 106spores/ml). Another four sterilized leaves were inoculated with 20 μL sterile water as a control. All plants were incubated in a chamber with 98% relative humidity at 25 ± 1°C. After 12 days, disease symptoms similar to the field were observed on leaves, whereas control plants remained healthy.P. capitalensiswas successfully reisolated only from the inoculated leaves and identified based on morphological characters. P. capitalensiscausedleafspotson various host plants around the world (Wikee et al. 2013), including on tea plants in China (Cheng et al. 2019) and oil palm in Malaysia (Nasehi et al. 2020), but it has not been reported on I. difengpi. Thus, this is the first report ofP. capitalensiscausingleafspoton I. difengpi. This study will provide an important reference for the control of the disease. The epidemiology ofthis disease should be investigated in further research.

Effect of climate and phenological stage on fungal endophytes community in Sorghum bicolor leaves

Endophytic fungi comprise a group of microorganisms of great diversity, which can be modulated by biotic and abiotic factors. The present study aims to understand how environmental factors, blooming and cultivars change the diversity of the endophytic fungal communities in sorghum leaves in two forest areas in Brazil. Our results showed that Sorghum bicolor has a rich and diverse community of endophytic fungi distributed in Ascomycota, Basidiomycota and Mucoromycota, with members of Dothideomycetes and Sordariomycetes as dominant groups. We found 581 endophytic fungi (filamentous and yeasts) identified in 55 species of 37 genera. A greater diversity and richness of endophytic fungi were found in sorghum leaves from the Atlantic Forest (diversity = H′ = 2.68 and richness = 46), differing significantly from the Caatinga forest (diversity = H′ = 2.51 and richness = 25). No difference in richness or diversity was observed between the two crops or between the phenological stages. We detected different species indicator for the two areas, nine taxa were associated with sorghum in the Atlantic Forest (Colletotrichum sp. 2, Epicoccum sorghinum, Curvularia sp., Colletotrichum sp. 1, Fusarium thapsinum, Nigrospora oryzae, Phyllosticta capitalensis, Fusarium oxysporum and Amesia nigricolor) and five taxa were indicators of sorghum in the Caatinga Forest (Meyerozyma sp., Talaromyces pinophilus, Metschnikowia sp., Rhodosporidiobolus sp. and Acremonium pinkertoniae). One species was an indicator of the cultivar IPA 2502 and no species were related to the cultivar IPA SF15. In relation to pre-blooming and post-blooming stages, two species were considered indicators for the former while six were considered for the latter. We observed that the number of filamentous fungi increases with the post-blooming stage and higher rainfall, while the diversity of endophytic yeasts was influenced by smaller rainfall and pre-blooming stage. Our data improves the understanding of factors that may influence the endophytic fungal community in ecologically and economically important grass species. The endophytic fungal diversity associated with sorghum is important to the mycodiversity estimation and may be useful in studies of biocontrol of pathogens and promotion of crops growth.

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.

Metabolites profiling and molecular docking identification of putative leads from endophytic Phyllosticta capitalensis as modulators of key druggable structural targets of rotavirus A

Rotavirus-induced gastroenteritis is a global health concern and the leading cause of death in low-income countries and children under the age of five. While rotavirus A (RVA) vaccines are available, there accessibility and efficacy remain challenging, especially in the third world countries. This has necessitated the need to develop therapeutics to manage RVA infection. This study investigated gas chromatography-mass spectrometry-derived Phyllosticta capitalensis metabolites as potential therapeutics against the key structural proteins [VP8* portion of the VP4 (spike protein), the VP7 (capsid protein), and the VP1 (RNA-dependent RNA polymerase)] of RVA using molecular docking and ADME approaches. The results revealed that four [lup-20(29)-en-one, A’-neogammacer-22(29)-ene, stigmasta-4,7,22-trien-3-α-ol, and fucosterol] of the 67 metabolites had favourable affinity for the three structural proteins and belonged to classes of metabolite of antiviral importance. Further analysis and pharmacokinetic profiling showed that the pentacyclic triterpenoids, lup-20(29)-en-one and A’-neogammacer-22(29)-ene, had the potential to be developed as antivirals against RVA infection and effort is underway in this direction.

Antimicrobial activity of n-hexane and ethyl acetate extracts from Candida tropicalis and Phyllosticta capitalensis fungal endophytes

Background ad objective: Among the notable achievements of the twentieth century was the discovery and identification of new drugs from plants against microbial infections. However, the discovery of novel drugs since then is inadequate due to emergence of resistant microbes. In an effort to discover novel drugs, the study aimed to investigate the antimicrobial activity of crude extracts from endophytic fungi isolated from Cnidoscolas aconitifolius and Ocimum suave. Methods: Following morphological characterization and initial screening for antimicrobial activity, isolates that had higher inhibition were genotypes by Sanger sequencing. Two isolates (Candida tropicalis from O. suave and Phyllosticta capitalensis from C. aconitifolius) were tested for antimicrobial activity against Escherichia coli and Staphylococcus aureus. Results: Overall, the range of crude extract concentration was from 152 mg/mL to 1353 mg/mL, and that of a zone of inhibition was from 7 to 21 mm. The lowest minimum inhibition concentration (19>MIC>9.5) was observed in Phyllosticta spp. extract against S. aureus. Conclusions: Findings of the present study have shown that endophytes isolated from medicinal plants can generate secondary metabolites with therapeutic applications. Therefore, further investigations are warranted to decipher the content and structure of bioactive compounds that may be associated with the antimicrobial activity of crude extracts.

First Report of Phyllosticta capitalensis Causing Black Freckle Disease on Rubus chingii in China.

Rubus chingii is used as an important traditional Chinese medicine, and belongs to the family Rosaceae. The fruit has multiple pharmacological activities, including antioxidant, anti-inflammatory, and improving cognitive impairment (Na Han et al. 2012). In June 2019, a new fungal infection was observed on the leaves of R. chingii in Qiandongnan Miao and Dong Autonomous Prefecture, Guizhou Province, China, forming small lesions with reddish-brown edges along leaf veins. Over 500 plants were surveyed, and nearly 20% of the plants were symptomatic. The diseased plants grew poorly and appeared stunted, and severely affected plants died. Five symptomatic leaves were randomly collected from the field and washed with tap water and distilled water successively. The edges of infected leaf tissue were cut into small pieces (4 to 5 mm2), surface sterilized with 70% ethanol for 30 s and 0.1% HgCl2 for 1 minute, and then rinsed three times in sterile distilled water (Chen et al. 2016). The same fungus was isolated from 41 pieces. The hyphae of a representative isolate were gray, the colony surface was granular, the edges were uneven and white, and the culture turned black over time with black spherical conidia. Conidia were nearly elliptical, unicellular, and each with a hyaline, unstable apical appendage, 3 to 10 µm long. The size of conidia was 10 to 18 μm in length and 4 to 8 μm in width. These morphological characteristics are consistent with those described for the fungus Phyllosticta capitalensis. (Wikee et al. 2013). For an accurate identification, genomic DNA of a representative isolate of the pathogen was extracted to amplify the internal transcribed spacer (ITS) region, the transcription elongation factor (tefa-1), and actin (ACT) genes with the ITS1/ITS4, EF1-728F/EF1-986R, and ACT-512F/ACT-783R (Cheng, L. L. ,et al. 2019), respectively. The ITS, tefa-1 and actin gene sequences were deposited in GenBank and assigned accession numbers MW308365, MW714380 and MW714381, respectively. BLAST search analysis of GenBank (NCBI) showed that the sequences had 100% similarity with those of Phyllosticta capitalensis (GenBank accession no. ITS, MN548091; tefa-1, MN958711; and ACT, MN565575). The pathogenicity of Phyllosticta capitalensis was verified using six healthy detached leaves from healthy R. chingii plants around 40 cm tall. A total of nine plants were used, and three leaves from each plant were artificially inoculated. Each wound was inoculated with conidial suspension (106 mL-1), while the control leaves were coated by sterile water. All the treated plants were covered with plastic bags for 2 days, incubated at 28ºC and 85% relative humidity, with a 12-hour photoperiod. After 15 days following inoculation, the injured leaves showed similar symptoms to the above-mentioned lesions, while the control and uninjured leaves were still healthy. P. capitalensis were reisolated from inoculated leaves, fulfilling Koch's postulates. P. capitalensis is an endophyte, widely distributed in various host plants in China. (Lu, J. M, et al. 2016). To the best of our known, this is the first report of black freckle disease caused by P. capitalensis on Rubus chingii in China. P. capitalensis is a destructive plant pathogen with an unusually broad host range and our findings will be useful for its management and for further research. The author(s) declare no conflict of interest.