Apical Appendages Research Articles

First Report of Pestalotiopsis lushanensis Causing Black Leaf Spot on Italian Ryegrass in China.

Italian ryegrass (Lolium multiflorum) is widely grown for forage in Southwest China. In December 2023, black leaf spots were observed on Italian ryegrass in a 10 hm2 field in Hongya County, Sichuan Province, China (29°52'11''°N, 103°16'16''°E). The disease developed sporadically with an incidence rate of < 1%. Leaves showed typical symptoms of small, black spots. For isolation, six samples (about 0.5 cm × 1 cm) were cut with a sterilized blade at the junction of diseased and healthy tissues from two different plants. They were surface disinfected in 75% ethanol for 40s, rinsed thrice in sterilized distilled water, air-dried, plated on potato dextrose agar (PDA), and incubated in the dark at 25°C for 3-7 days. A Pestalotiopsis-like fungus with similar colony characteristics was consistently isolated from all samples. Pure cultures were obtained with the single-spore technique (Cai et al. 2009), and two strains (LJH17 and LJH18) were selected for further study. After 5 days in the dark at 25°C, colonies on PDA were cottony, whitish, 70-78 mm in diameter, with an entire edge. The reverse side of the colonies appeared white to light yellow. Conidia were fusoid, straight to slightly curved, 4-septate, with 2-4 (mostly 3) tubular apical appendages, measuring 17.3 to 25 × 6.1 to 9.9 μm (mean ± SD = 20.7 ± 2.3 × 7.9 ± 0.7 µm; n = 50; LJH17). For further identification, the internal transcribed spacer regions 1 and 2 and 5.8S nuclear ribosomal RNA (ITS), partial β-tubulin (tub2) and translation elongation factor 1-α (tef1) genes from the two pure strains GMCC000083 (=LJH17; GMCC, Grassland Microbiome Culture Collection) and LJH18 were amplified with the primer pairs ITS1/ITS4 (White et al. 1990), T1/Bt2b (Glass and Donaldson 1995; O'Donnell and Cigelnik 1997), and EF1-728F/EF2 (Carbone and Kohn 1999; O'Donnell et al. 1998). Sequences were deposited in GenBank (PP779763, PP781949, and PP781950 for LJH17; and PQ013725, PQ015095, and PQ015096 for LJH18). BLAST analysis showed 100% identity across all three segments with ex-type isolate LC4344 of Pestalotiopsis lushanensis (Liu et al. 2017). A randomized accelerated maximum likelihood (RAxML) phylogenetic tree based on concatenated ITS, tub2, and tef1 alignments showed that our two strains clustered with the previously reported P. lushanensis strains ((Liu et al. 2017; Jiang et al. 2022) (Fig. S1F). Eight-week-old healthy plants were grown from Italian ryegrass seeds in pots, with three plants per pot. Following our previous spray inoculation procedures (Xue et al. 2020): fresh 5-day-old mycelia from each strain were prepared as mycelial suspensions at about 2.5 × 104 CFU/ml, and conidial suspensions were adjusted to about 2 × 105 conidia/ml. For each strain (LJH17 and LJH18), five pots were separately spray-inoculated with conidial and mycelial suspensions (about 2 ml/pot). Five pots served as non-inoculated controls were sprayed with sterilized distilled water. All pots were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse at 23-29°C. After incubation for 15 days, both conidial and mycelial suspensions produced black spots on the plants (Fig. S1), resembling those previously observed in the field. No symptoms were observed in the control plants. Pathogenicity tests were conducted thrice using the same method. The same fungus was reisolated from the lesions and confirmed via the morphological and molecular methods described above, thus fulfilling Koch' s postulates. To our knowledge, this is the first report of P. lushanensis causing black leaf spot on Italian ryegrass in China or worldwide. This finding may aid in diagnosing, identifying the pathogen, and controlling the disease on Italian ryegrass.

First Report of Leaf Blight of Ginkgo biloba L. Caused by Neopestalotiopsis clavispora G.F. Atk. in Sichuan, China.

Ginkgo (Ginkgo biloba L.), the oldest existing tree species in the world, is an important ornamental and medicinal plant, widely planted in China. In October 2022, a new leaf blight disease was observed in Chengdu city (30°05'to 31°26'N, 102°54'to 104°53'E). Disease incidence averaged 82.5% across five foci. The typical symptomatology begins when leaf margins turn yellow and small black spots appear at the edge of the leaf, chlorotic areas turn brown, dry and deformed. Gradually, the necrotic lesions spreads to the middle of the leaf and eventually the whole leaf falls off. Infected tissues from ten leaves were cut into small pieces (5 × 5 mm); surface sterilized for 30 s in 3% sodium hypochlorite; 60 s in 75% ethanol; rinsed three times in sterile water; placed onto potato dextrose agar (PDA) amended with streptomycin sulfate (50 μg/mL); and incubated at 25°C for 3 to 8 days. A hyphae was removed from the edge of the fungal colony and placed onto potato dextrose agar (PDA) plates. After incubation at 25℃ with a 12-hour light/dark cycle for 8 days, the colony diameter reached 77.5 to 81.5 mm. Colonies grown on PDA were white, cotton, flocculent, undulating on the surface, dense in aerial hyphae and light yellow on the back. Black pycnidia formed superficially, scattered over the PDA, following two weeks of incubation. Pycnidia contained sticky black conidia. The spores were were spindle shaped, with five cells, and four septations measuring 20.9 to 34.8 µm × 6.8 to 8.8 µm (avg. 28.4 × 7.6 µm; n=40). The three median cells were versicolored, typically two dark brown cells and one light brown cell, whereas the basal and apical cells were hyaline. Conidia had a single basal appendage (2.87 to 4.1 µm long; n = 40) and two to three apical appendages (18.3 to 29.1 µm long; n = 40). Based on colony and conidial morphology, the isolate was identified as N. clavispora (Maharachchikumbura et al. 2014). The partial sequence of the internal transcribed spacer (ITS), β-tubulin gene (TUB2), and translation elongation factor subunit 1-a gene (TEF1) were amplified and sequenced using the universal primer pairs ITS1/ITS4(Zhang et al. 2022), BT2A/BT2B (Li Yuan et al. 2022), and EF1-526F/EF1-1567R (Maharachchikumbura et al. 2012), respectively. Sequences of representative isolate LQYX were deposited in GenBank (ITS: OQ152504, TUB: OQ168328, and TEF1: OQ168329). BLAST results indicated that the ITS, TUB, and TEF1-α sequences showed 99 to 100% identity with N. clavispora sequences at NCBI (GenBank MG729689, MG740735, and MG740758). Identification was confirmed by Bayesian inference using Mr. Bayes. Next, inoculations were conducted on leaves of ten G. biloba in the field to verify the pathogenicity of LQYX. Ten healthy leaves of each plant were surface sterilized with 75% ethanol, and the wound was rubbed out on the leaf edge on the sterilized sanding paper. A conidia suspension (1 × 107 ml-1) was sprayed on the leaves, aseptic water was used as the control, and the transparent plastic bag was used to maintain relative humidity. After 14 days (26 ℃, 14 hours light / 10 hours dark), the inoculated leaves had similar symptoms as the original diseased plants, whereas controls were asymptomatic. The N. clavispora was re-isolated from the infected leaves and identified by morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times with similar results, confirming Koch's postulates. To our knowledge, this is the first report of leaf blight of G. biloba caused by N. clavispora in China, which has greatly affected the appearance of the city and should be further studied. This report can help identify this disease and further develop effective control measures.

First report of Neopestalotiopsis clavispora Causing guava scab in China.

Psidium guajava L. is widely cultivated in southern China. In May 2021, guava scab on cv. Zhenzhu was observed in Zhanjiang (21.18° N, 110.21° E), Guangdong province, China. Guavascab was corky with ovoid or round lesions on the surfaces of green fruits. Gradually the lesions sunk. Disease incidence was estimated as 85% in 500 investigated plants in about 50 ha. Twenty diseased fruits were collected from twenty trees in the field. From each fruit the margin of the diseased tissues was cut into 2 mm × 2 mm pieces; surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, successively; and rinsed thrice with sterile water. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 ℃. Thirty-four isolates were obtained. Single-spore isolation method (Liu et al. 2021) was used to recover pure cultures of three isolates (PGNC-1, PGNC-2, and PGNC-3) . The colonies were initially white with cottony aerial mycelium at 7 days on PDA. Then, these colonies form black acervular conidiomata at 10 days. Conidia were clavate to fusiform, four-septate, straight or slightly curved, and measured 15.8 to 21.2 µm × 4.5 to 6.5 µm (n = 40). The three median cells were versicolored, whereas the basal and apical cells were hyaline. Conidia had a single basal appendage (4.5 to 5.5 µm long; n = 40) and three apical appendages (19.2 to 24.5 µm long; n = 40). The morphological characteristics of the isolates were consistent with the description of Neopestalotiopsisclavispora (Maharachchikumbura et al. 2012). Molecular identification was performed using PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012). Sequences were generated from the isolates using primers for the rDNA ITS (ITS1/ITS4), TEF1-α (EF1-728F/EF1-986R), and β-tubulin (T1/βt2b) loci (Maharachchikumbura et al. 2012). The sequences of the isolates were submitted to GenBank (ITS, OQ996557 to OQ996559; TEF, OR101037 to OR101039; β-tubulin, OR100971 to OR100973). The sequences of the isolates were 100% identical to the type strain MFLUCC12-0281 (accession nos. JX398979, JX399014, and JX399045) through BLAST analysis. The isolates clustered with N. clavispora (MFLUCC12-0280 and MFLUCC12-0281). N. clavispora and Pestalotiopsis clavispora are synonyms. The pathogenicity was tested in vivo. Plants (cv. Zhenzhu) were grown ( 3 years old) in a quarantine orchard at 25℃ to 32℃ with 60 to 80% relative humidity in May 2022. Disease-free green fruits were inoculated. Sterile cotton balls were immersed in the spore suspension (1 × 105 per mL) and sterile distilled water (control) for about 15 s before they were fixed on the wounded fruits with transparent tape. Five fruits on one plant per isolate were inoculated. Five fruits on one plant severed as control. The test was performed thrice. Disease symptoms were found on the inoculated fruits after 20 days, whereas the controls remained healthy. The pathogen was re-isolated from infected fruits and was phenotypically identical to the original isolates thus fulfilling Koch's postulates. Neopestalotiopsis or Pestalotiopsis spp. were reported to be the causal agents of guava scab in Colombia and in Hawaii (Keith et al. 2006; Solarte et al. 2018). N. clavispora has been reported to cause disease in a broad range of hosts (Ge et al. 2009; Chen et al. 2018), but not in guava. This is the first report of N. clavispora causing guava scab in China. There would be no harvest if this disease is left unmanaged.

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.

Appendopyricularia guttulata sp. nov., a lignicolous freshwater fungus from Dianchi Lake in Yunnan, China

During an investigation into species diversity of lignicolous freshwater fungi from the plateau lakes in Yunnan Province, China, a hyphomycetous fungus was collected from Dianchi Lake. The new isolate was placed in Appendopyricularia as a sister taxon of Appendopyricularia juncicola within Barbatosphaeriaceae based on the phylogenetic analysis of combined ITS and LSU sequence data. The fungus is characterized by acrogenous, fusoid, guttulate conidia that are 3–4 septate and have a flexuous central apical appendage. It exhibits more pronounced guttulate appendages and more prominent denticle sporulation structure. Therefore, Appendopyricularia guttulata is introduced as a new species based on morphological characteristics and phylogenetic evidence. The detailed description and illustrations of the new species are provided, as well as the morphological comparison with similar taxa.

First report of leaf spot on Daphniphyllum macropodum caused by Neopestalotiopsis clavispora in China.

Daphniphyllum macropodum Miq., an evergreen arbor, is widely cultivated in southern China for its ornamental and medicinal value (Su et al. 2013). In October 2019, a severe leaf spot was observed on D. macropodum in Jinggangshan National Nature Reserve in Ji'an city, Jiangxi, China (114°06'23″E, 26°32'25″N). The plants were about 15 years old, and the disease incidence was estimated to be 15% (4/26 plants). The disease primarily appeared as small black spots on the leaves. At the late stage, the spots enlarged and coalesced into regular or irregular gray necrotic lesions with dark margins. We collected five samples per plant and total 20 samples were collected to isolated the pathogen strains. The margin of the diseased tissues was cut into 5 mm × 5 mm pieces; surface disinfected with 70% ethanol and 2% NaOCl for 30 s and 60 s, respectively; and rinsed thrice with sterile water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C in the dark. Pure cultures were obtained by single-spore isolation method, and the representative isolates, JRM3, JRM6, and JRM8 were used for morphological studies and phylogenetic analyses. The colonies of three isolates grown on PDA were white, cottony, and flocculent, contained undulate edges with dense aerial mycelium on the surface at 25 °C. Conidiomata was black conidial masses on PDA. Conidia were 5-celled, clavate to fusiform, smooth, 19.3 to 24.4 long × 6.1 to 8.6 μm wide (n = 50). The 3 median cells were dark brown to olivaceous, the central cell was darker than the other 2 cells, and the basal and apical cells were hyaline. All conidia developed one basal appendage (3.4 to 8.3 μm long; n = 50), and 2 to 3 apical appendages (18 to 32 μm long; n = 50), filiform. The morphological characteristics of the isolates are comparable with those of the genus Neopestalotiopsis (Maharachchikumbura et al. 2014). The internal transcribed spacer (ITS) regions, β-tubulin 2 (TUB2) and translation elongation factor 1-alpha (TEF1-α) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, T1/Bt-2b, EF1-728F/EF-2 (Maharachchikumbura et al. 2014), respectively. The sequences of the isolates were submitted to GenBank (ITS, OQ372202 to OQ372204; TUB2, OQ390129 to OQ390131; TEF1-α, OQ390126 to OQ390128). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed JRM3, JRM6, and JRM8 in the clade of N. clavispora. Based on the multi-locus phylogeny and morphology, three isolates were identified as N. clavispora. To confirm pathogenicity, eight healthy 10-year-old D. macropodum plants growing in the field were chosen, and 4 leaves per plant were wounded with a sterile needle and inoculated with 10 μL conidial suspension per leaf (106 conidia/ml). Eight plants inoculated with sterile water were used as control. All the inoculated leaves were covered with plastic bags to maintian a humidity environment for 2 days. The leaves inoculated with conidial suspension showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 10 days. The same fungus were re-isolated from the lesions, whereas no fungus was isolated from control leaves. N. clavispora was determined as the pathogen of a variety of plant diseases, including Kadsura coccinea (Xie et al. 2018), Dendrobium officinale (Cao et al. 2022), Macadamia integrifolia (Santos et al. 2019). However, this is the first report of N. clavispora infecting D. macropodum in China. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

First Report of Phyllosticta capitalensis Causing Leaf spot in Hymenocallis littoralis in China.

Hymenocallis littoralis (Jacq.) Salisb. is a common ornamental plant in China. In November 2021, leaf spots were observed on H. littoralis in a public garden in Zhanjiang, Guangdong Province, China (21°17'25″N, 110°18'12″E). Disease incidence was around 60% (n = 100 investigated plants from about 1 ha). Leaf symptoms were round spots with collapse centers, surrounded by yellow halos. Ten symptomatic leaves from 10 plants were sampled. The margins of the samples were cut into 2 mm × 2 mm pieces. The surfaces were disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Thereafter, the samples were rinsed thrice in sterile water, placed on PDA, and incubated at 28 °C in dark. Pure cultures were obtained by transferring hyphal tips to new PDA plates. Twenty pure cultures were obtained. Single-spore isolation method (Liu et al. 2021) was used to recover the cultures of three isolates (HPC-1, HPC-2, and HPC-3). Colonies of the isolates were dark green with a granular surface, and irregular white (later turning black) edge. Pycnidia were black, globose and 96 -140 μm in diameter. Conidia were single-celled, oval, 7.5 to 13.5 × 4.0 to 7.5 µm (n= 40), with a single apical appendage. Morphological characteristics of the isolates were consistent with the description ofPhyllostictacapitalensis(Wikee et al. 2013). Molecular identification was performed using PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012). The internal transcribed spacer (ITS) region, translation elongation factor (TEF1), actin (ACT), and glyceradehyde-3-phosphate dehydrogenase (GAPDH) were amplified using primers ITS1/ITS4, EF1-728F/EF1-986R (Druzhinina et al. 2005), ACT-512F/ACT-783R, and Gpd1-LM/Gpd2-LM (Wikee et al. 2013), respectively. Sequences were deposited in GenBank with accession numbers OM654570 - OM654572 forITS, OM831376 - OM831378 fortef1-α, OM831346 - OM831348 forACT, and OM831364 - OM831366 for GAPDH. BLASTn analysis showed that these sequences were 99 to 100% similarity with those of P.capitalensis(ITS, FJ538339; TEF1, FJ538397; ACT, FJ538455; and GAPDH, JF343723). Besides, a phylogenetic tree was generated on the basis of the concatenated data from the sequences of ITS, TEF1, ACT, and GAPDH that nested within the clade containing P.capitalensis (CBS 117118, CPC20510,CPC20267, and CPC18848). From the combination of the morphological and molecular characteristics, the isolates were determined to beP.capitalensis. Pathogenicity testing was performed in a greenhouse with 80% relative humidity at 25 to 30°C. Ten healthy plants of H. littoralis (2 month old with 4 leaves) were grown in pots with one plant in each pot. Three leaves on one plant per isolate were inoculated with three mycelial plugs obtained from 7-day cultures, totaling five plants. Five plants treated with PDA plugs served as the controls. Wet cotton balls were fixed on the leaves with transparent tape for five days to keep it from drying out. The test was conducted three times. After 15 days, similar symptoms were observed in the inoculated leaves as in the garden, whereas control leaves remained asymptomatic and P.capitalensiswas successfully re-isolated from the inoculated leaves. Previously, P.capitalensishas been reported to cause leaf spot disease of various host plants around the world (Wikee et al. 2013). However, to our knowledge, this is the first report of leaf spot caused by P.capitalensison H. littoralis in China. This study provides an important reference for the control of the disease.

Fossil seeds of Passiflora L.: An Oligocene record of a new species and a Pleistocene record of a modern species from the Gulf of Mexico Coastal Plain

We describe two small, compressed seeds from the Gulf of Mexico Coastal Plain that display a reticulate-foveolate seed coat pattern as species of Passiflora subgenus Passiflora. One was obtained from the basal layer of the Catahoula Formation in Wayne County, Mississippi, and is late Oligocene in age (25–23.8 Ma), supported by planktonic foraminferal and nannofossil zones. The other is from a Pleistocene river terrace (luminescent dating of 85–82 ka) on the banks of the Mobile River in Alabama. The Oligocene seed is tridentate (consisting of a central apical appendage and well-displayed lateral protrusions to the sides of this appendage), symmetrical, and has a palisade seed coat structure with ruminate endosperm. Its characters allow tentative placement within supersection Laurifolia and series Tiliifolia. Extensive comparisons with seeds of extant species of Passiflora subgenus Passiflora serve as the basis for recognizing it as a new species, Passiflora axsmithii sp. nov. The Pleistocene seed is obovate, slightly asymmetrical, has a small crown around the base of the apical appendage, and insubstantial lateral projections. As the Pleistocene seed is geologically young and fits the description of seeds produced by the native extant species Passiflora incarnata (supersection Passiflora, series Passiflora), we consider it very likely to be a representative of that species. The Oligocene seed is the earliest reliable record, thus far, of genus Passiflora. The Pleistocene seed is likely the earliest record, to date, of the extant species P. incarnata.

Phyllosticta taxa (Botryosphaeriales, Phyllostictaceae) from northern Thailand and southern European Russia: New host and geographical records

Phyllosticta is a cosmopolitan group of fungi found on various host plants, occurring as pathogens, endophytes and saprobes. Diseases caused by Phyllosticta commonly include leaf and fruit spots that can affect economically important plants. The genus is characterized mainly by aseptate and hyaline conidia and ascospores. Its conidia are surrounded by a mucilaginous sheath, with a single mucoid apical appendage while ascospores exhibit a mucoid cap at both ends. Given that many Phyllosticta taxa are cryptic and share similar morphologies, it is arduous to depict taxonomically relevant characters solely on the basis of morphological or ecological features. Coupled with morphological description, multi-locus phylogenetic analyses of species comprising complexes are used to broadly describe this genus and understand species boundaries. Despite several published taxonomic revisions and enumerations of Phyllosticta species, there is still considerable confusion when identifying these taxa. Herein, we establish three new host records for P. capitalensis, and one new geographical record for P. musaechinensis in Thailand, and one new host and geographical record for P. citribrasiliensis in Russia. We provide an updated phylogenetic tree based on several gene markers: ITS, LSU, ACT, GAPDH, RPB2, and TEF-1α. This includes all Phyllosticta species for which sequence data is available. Six individual Phyllosticta species complexes are accepted. In addition, we synonymize P. ericarum with P. citribrasiliensis.

First report of Truncatella angustata causing leaf blight on Thuja plicata Donn ex D. Don in Canada.

Western redcedar (Thuja plicata Donn ex D. Don) is one of the most important commercial tree species in British Columbia, generates more than $1 billion in economic activity annually and about 8-10 million trees are planted in reforestation efforts (Gregory et al. 2018). It has been selected as the provincial tree of British Columbia (BC) because of its tremendous economic, ecoogical and cultural value. However, foliar diseases such as leaf blights have serious impact on redcedar growth and may cause significant loss of tree volume (Russell, 2007). Our 2014 - 2015 surveys of western redcedar forests in coastal areas of BC indicated high incidence of a distinctive type of blight. We observed the incidence of this disease on more than 80% of western redcedar (approximately 493) trees from late May to early December. Early symptoms appeared as circular to oval, brownish to black spots (2-3 mm), 1-5 spots per branch tip, scattered at the tip margins. Sequentially, the spots enlarged and developed into necrotic lesions on both young and old leaves. More than 50 symptomatic leaves from 10 different trees were collected and rinsed in distilled water then surface-sterilized with three times washing in Tween 20 (%5 solution) for 2 minutes (each time) and %70 ethanol for 30 second (3 times repeat). Tissues from under lesions were placed on MEA (Malt Extract Agar; Phyto Tech® labs-Product ID: M498) and PDA (Potato Dextrose Agar; Phyto Tech® Labs-Product ID: P772). The plates were incubated at 21°C in the dark. They developed distinct dull white to brown, cottony colonies with each black acervuli approximately 450-500µm. The isolates produced fusiform conidia with four cells. They didn't have any distinct color. The conidiophore size was approximately 23-24 x 2-3 µm with mostly hyaline to light brown color, branched and conidiogenous was hyaline and not branched and simple. The spore size was approximately 15-20μm by 7-10μm with three transverse septa and endogenous papillae with hyaline apical appendages. Next, we collected spores and replated them on fresh MEA media culture and placed back in the incubator to produce pure cultures. We studied conidia from leaves of trees mentioned above using light and electron microscopy using Hitachi S-3500N Scanning Electron Microscope (Noshad et al. 2023). After morphological study, further identification to the species level conducted using Zambounis and Wenneker's approach (Zambounis 2019; Wenneker,2017). Genomic DNA from two single-spore isolates were isolated and sequenced. Sequences of ITS (Internal Transcribed Spacer) region amplified using primers ITS1/ITS4 and sequenced. Final sequences were deposited in Genbank and published (accession numbers OP086244 and OP086251). Blast analysis of these sequences showed 99% and 99% resemblances with T. angustata sequence (Sutton 1980). To verify its pathogenicity, we performed a comprehensive pathogenicity test to fulfill Koch's postulates. We collected their distinctive spores in an aseptic environment and standardized them (5000/ml) using a haemocytometer. Then we inoculated 100 western redcedar seedlings (three years old) by injecting standardized spore suspension solution (inoculum) using ultra-fine 0.3ml, 31G, 8mm syringes (approximately 0.1ml per inoculation site). Ten positive control seedlings were inoculated with distilled water and ten negative control seedlings were not inoculated at all. All inoculated (experimental) seedlings demonstrated same symptoms (black spots and characteristic spores) after eight weeks. None of the control seedlings showed any similar symptoms. In the next stage, we isolated and cultured spores from inoculated seedlings and studied them. The identity of reisolates confirmed using DNA sequencing. We used these spores for our next set of disease screening which was successful again. We identified Truncatella angustata (Pers.) Hughes as the causal agent for shoot-tip blight (STB) on western redcedar by examining morphological and molecular characteristics of the pathogen. This is the first report of T. angustata as a primary pathogen on western redcedar in British Columbia, Canada.

Tetraploa wurfbainiae sp. nov. (Tetraplosphaeriaceae, Pleosporales) isolated from Wurfbainia villosa in Guangdong, China

ABSTRACT During a survey for microfungi associated with traditional medicinal plants in Guangdong Province, China, an interesting species in the Tetraplosphaeriaceae was isolated from the dead stem of Wurfbainia villosa (Zingiberaceae). The new collection is characterised by brown to dark brown, cylindrical to ovate, four-columned conidia, mostly with four straight or slightly flexuous, smooth, and long apical appendages. The phylogenetic analysis of the combined dataset of internal transcribed spacer (ITS), large subunit nuclear rDNA (LSU), the partial translation elongation factor 1-α (tef1-α), and β-tubulin (tub2) further indicated that the new collection forms a distinct branch in Tetraploa and clusters as a sister taxon with T. dwibahubeeja. Thus, we introduce a new species, Tetraploa wurfbainiae, based on distinct morphology and multi-locus phylogeny, accompanied by a description, illustration, and morphological comparison with related taxa. This is the first report of Tetraploa species on the medicinal plant W. villosa. This study enhances our understanding of the fungal species associated with the medicinal plant W. villosa.

Volvariella lanata (Pluteaceae, Agaricales), a new species from the Middle Volga Region, Russia

One new species of Volvariella (V. lanata) is described based on a combination of morphological and molecular evidence. The species is characterized by rather large basidiomata with grey-brown, fibrillose to pubescent pileus, pubescent stipe and brownish trilobate volva, cheilocystidia with long apical appendages, utriform or fusiform pleurocystidia, and basidiospores 5.9–7.4 × 3.7–5.0 µm. Sequences of nrITS and nrLSU of the studied specimens were generated, and the phylogenetic analyses were performed with Maximum likelihood and Bayesian inference methods for nrITS+nrLSU dataset. The analyses showed that the new species is clustered within the genus Volvariella, forming a distinct phylogenetic lineage with V. krizii as its sister taxon.

PAX6 protein in neuromasts of the lateral line system of salamanders (Eurycea).

PAX6 is well known as a transcription factor that drives eye development in animals as widely divergent as flies and mammals. In addition to its localization in eyes, PAX6 expression has been reported in the central nervous system, the pancreas, testes, Merkel cells, nasal epithelium, developing cells of the inner ear, and embryonic submandibular salivary gland. Here we show that PAX6 also appears to be present in the mechanosensory neuromasts of the lateral line system in paedomorphic salamanders of the genus Eurycea. Using immunohistochemistry and confocal microscopy to examine a limited number of larvae of two species, listed by the United States of America's federal government as threatened (E. nana) or endangered (E. rathbuni), we found that anti-PAX6 antibody labeled structures that were extranuclear, and labeling was most intense in the apical appendages of the hair cells of the neuromast. This extranuclear localization raises the possibility of an as yet undescribed function for PAX6 as a cytoskeleton-associated protein.

First report of Neopestalotiopsis clavispora causing leaf spot on Phoebe bournei in China.

Phoebe bournei (Hemsl.) Yang is a typical evergreen broadleaf species widely distributed in subtropical China for its ornamental and economic value (Zhang et al. 2021). The wood of P. bournei is considered a good material for architectural decoration and furniture (Li et al. 2018). In June 2020, leaf spot symptoms were observed in Dexing (28°41'22.056″N, 115°51'52.524″E), Jiangxi province, China. Initial disease symptoms were small brown spots on the leaves. Then, the spots enlarged and coalesced into regular or irregular dark brown necrotic lesions with dark margins. Disease incidence in the field in Dexing was estimated 25%. Leaf pieces (5 × 5 mm) from the lesion borders were surface-sterilized in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C under a 14/10 h light/dark cycle for 4 days. Pure cultures were obtained by monosporic isolation, and the representative isolates, JX-N2, JX-N7, and JX-N11 were used for morphological studies and phylogenetic analyses. The colonies of three isolates grown on PDA were white, cottony, and flocculent, contained undulate edges with dense aerial mycelium on the surface. Conidia were 5-celled, clavate to fusiform, smooth, 18.7-24.6 × 5.9-8.8 μm (n = 100). The 3 median cells were dark brown to olivaceous, central cell was darker than other 2 cells, and the basal and apical cells were hyaline. All conidia developed one basal appendage (3.4-8.3 μm long; n = 100), and 2-3 apical appendages (17-30 μm long; n = 100), filiform. Morphological features were similar to Neopestalotiopsis sp. (Maharachchikumbura et al. 2014). The internal transcribed spacer (ITS) regions, β-tubulin 2 (TUB2) and translation elongation factor 1-alpha (TEF1-α) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, T1/Bt-2b, EF1-728F/EF-2 (Maharachchikumbura et al. 2014), respectively. All sequences were deposited into GenBank (ITS, OQ355048 - OQ355050; TUB2, OQ357665 - OQ357667; TEF1-α, OQ362987 - OQ362989). A maximum likelihood and Bayesian posterior probability-based phylogenetic analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (ITS, TUB2, TEF1-α) placed JX-N2, JX-N7, and JX-N11 in the clade of N. clavispora. Based on the multi-locus phylogeny and morphology, the representative isolates were identified as N. clavispora. The pathogenicity of three isolates were tested on six 9-year-old P. bournei plants, which were grown in the field. Three leaves per plant were wounded with a sterile needle (Φ=0.5 mm) and inoculated with 20 μL conidial suspension per leaf (106 conidia/mL). Another six control plants were inoculated with sterile water. Each leaf was covered with plastic bags to keep a humidity environment for 2 days. All the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 9 days. N. clavispora was reisolated from the lesions, whereas no fungus was isolated from control leaves. N. clavispora can cause leaf diseases in a variety of hosts, including Machilus thunbergii (Wang et al. 2019), Fragaria × ananassa (Shi et al. 2022), Taxus media (Li et al. 2022). However, this is the first report of N. clavispora infecting P. bournei in China. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

First Report of Pestalotiopsis trachicarpicola Causing Apothecium Wither on Cultivated Morchella sextelata in Sichuan, China.

Morel (Morchella sextelata) is a valuable medicinal and edible mushroom, but the wild yield is seriously insufficient, so several artificial varieties have been developed to alleviate the shortage of wild yield. However, in 2020-2022, apothecium wither symptoms were observed in Nanchong, Sichuan. A total of 30.80% of the morels in the planting base (1.3 km2) showed these symptoms. The initial symptoms were slight white lesions on the surface of apothecium, then the mycelia gradually invaded the interior of the fruiting body, and eventually leading to black and wilt apothecium (Fig. 1a). Fifteen symptomatic morels were collected and ten isolates were obtained using the single spore isolation technique developed by Chomnunti et al. (2014). The morphological characteristics of the ten isolates were similar, which showed dense aerial white mycelia colony texture on PDA, and later forming concentric black mucus (Fig. 1c). The reverse side was yellow (Fig. 1d). The acervulus was floral-shaped and discrete, with smooth walls and measured 120.0 to 400.5 × 15.5 to 40.0 μm (n=10) (Fig. 1e); the conidia were fusiform and hyaline, 21.0 to 28.6 × 6.0 to 7.6 μm in size (n=50), each contained five cells. The apical cell was hyaline, conic and with 2 to 4 tubular apical appendages on the top. The three intermediate cells were brown to olivaceous, doliform to cylindrical, constricted at septa. The basal cell was conic to acute (Fig. 1f). The morphological characteristics were consistent with the published description of Pestalotiopsis trachicarpicola (Maharachchikumbura et al., 2012). PCR was performed with primers ITS1/ITS4 for internal transcribed spacer (ITS) (White et al., 1990), BT2A/BT2B for β-tubulin gene (TUB) (Glass and Donaldson, 1995), and EF1-526F/EF1-1567R for translation elongation factor 1-alpha (TEF-1α) (Roger et al., 1999). The pairwise alignments of ITS, TUB, and TEF-1α sequences was nearly 100% identical to P. trachicarpicola with GenBank accession numbers MT889666.1 (579/585 bp, 99%), MT884145.1 (445/450 bp, 99%), and MW149930.1 (946/958 bp, 99%), respectively. The resulting sequences were deposited in GenBank (Accession no. ITS: OL362082; TUB: OL828342; and TEF-1α: OL905009). Phylogenetic analysis performed with maximum likelihood method used MEGA 7.0 (1000 bootstrap replications) classified WLM5 into the P. trachicarpicola clade (Fig. 2), so we finally confirmed the identity of WLM5 as P. trachicarpicola. To fulfill Koch's postulates, twenty morels were surface disinfected with 2% sodium hypochlorite and then artificially wounded (diameter of 0.5 mm) prior to inoculation with 200 μL conidial suspension (105 conidia/mL), while an equal amount of sterile distilled water was applied to controls. After 4 days, the inoculated fruiting bodies showed symptoms consistent with field infection (Fig. 1b) and P. trachicarpicola was re-isolated using the same protocol, while the control remained asymptomatic. This first report of P. trachicarpicola causing apothecium wither on morel will help develop robust disease management strategies against this emerging fungal pathogen.

First Report of Neopestalotiopsis clavispora causing Leaf Spot on Coconut Seedling in Malaysia.

Coconut (Cocos nucifera) is a high economic value cash crop in Malaysia. In December 2021, irregular spots with dotted rust-like appearance were observed mainly on the tip of the leaves of MATAG variety coconut seedlings at the nursery in Perak state. More than 90% of the coconut seedlings surveyed were infected with leaf spot symptoms. These symptoms could bring huge economic losses due to the downgrade value of the seedlings. 15 symptomatic leaves were obtained from the nursery, 10 mm2 of cut leaves were disinfected with 10% sodium hypochlorite for 10 minutes and rinsed with sterile distilled water before plated on potato dextrose agar (PDA). A total of 4 single-spore isolates were obtained and were observed morphologically. The isolates had white cotton-like appearance with undulate edge. Black acervuli were seen after 7 days of incubation at 26 °C. The conidia were fusiform and contained five cells with four septate and three versicolor cells in between the apical and basal cell. The conidia were 17.2 µm long and 5.9 µm wide (n=30). Conidia consisted of two to three apical appendages and one basal appendage. These morphological characters were consistent with the original description of Neopestalotiopsis clavispora (Santos et al., 2019; Abbas et al., 2022). Species identification was done by amplifying internal transcribed spacer (ITS) region using primers ITS 4 and ITS 5 (White et al., 1990) and beta-tubulin (TUB2) using primers Bt2a and Bt2b (Glass & Donaldson et al., 1995) of the representative isolate LKR1, then sequenced. The 488 bp ITS and 409 bp TUB2 sequences were deposited in GenBank under the accession numbers ON844193 and OP004810, respectively. Isolate LKR1 shares 99.8% identity with the ITS sequence (MH860736.1) of the reference pathogenic N. clavispora strain CBS:447.73 and 100% identity with the TUB2 sequence (KM199443.1) of the reference pathogenic N. clavispora strain CBS 447.73. The phylogenetic analysis confirmed that the isolate LKR1 belonged to N. clavispora when a supported clade is formed with 98% and 94% bootstrap support for ITS and TUB2 respectively with other related N. clavispora. Pathogenicity test was conducted by using five replicates of 8 month old seedlings, they were incubated under greenhouse condition and were watered daily. The leaves of the seedlings were injured with sterile needles and were sprayed with conidial suspension (1 x 10^6 conidia/ml). The control plants were also injured but sprayed with sterile distilled water. After a month, signature symptoms of spots on the leaves appear but none on the control seedling. N. clavispora was successfully re-isolated only from the inoculated symptomatic leaves and identified morphologically. No fungus was re-isolated from the control seedlings. The result was consistent even after repeating the test one more time. N. clavispora has been reported causing leaf spot on Macadamia integrifolia (Santos et al., 2019), Phoenix dactylifera L. (Basavand et al., 2020) and Musa acuminata (Qi et al., 2022). N. clavispora has also been reported causing rust-like appearance of leaves on strawberry (Fragaria × ananassa Duch.) (Obregón et al., 2018). To our knowledge, this is the first report of N. clavispora causing leaf spot disease on coconut seedlings in Malaysia. Through the identification of N. clavispora as the causal agent of leaf spot on coconut, this can help coconut growers to tackle the disease problem earlier thus, preventing the disease from spreading until the adult phase.

First report of Neopestalotiopsis clavispora causing leaf blight on Aegicerascorniculatum in China.

Aegicerascorniculatum is an important mangrove plant that mainly grows in tropical and subtropical regions. Ithasimportantecological,economicandsocialbenefits (Bandaranayake 1998). In April 2021, a leaf disease on A.corniculatum was observed in Zhanjiang (21.21° N, 110.41° E), Guangdong province, China. Diseaseincidenceandseverity were 15%and 20% (n = 100 investigated plants), respectively. The disease symptoms on leaves primarily appeared as small brown spots, then enlarged and coalesced into regular or irregular gray necrotic lesions with dark margins. At the late stage of symptom development, black acervuli appeared on the necrotic lesions. Ten symptomatic leaves from 10 plants were collected. Small pieces of tissue (4 × 4 mm) were cut from lesion borders and were surfaced disinfected in 75% ethanol for 30 s, followed by 1 min in 1% NaClO, rinsed three times with sterile water, plated on potato dextrose agar (PDA), and incubated at 28°C. After 7 days, a total of 10 fungal isolates with 100% isolation frequency were obtained and three representative strains (THS-1, THS-2, and THS-3) were used for morphological and molecular characterization.Colonies were white with cottony aerial mycelium and irregular margins. Black viscous acervuli were scattered on the colony surface 10 days after incubation. Conidia were spindle shaped, five cells, four septa, 18.77-28.70 × 4.53-6.80μm (mean 23.13 × 5.14μm) in size (n = 50). Basal and apical cells were colorless while the three medium cells were dark brown and lightly versicolor. All conidia had one basal appendage of 3.51-7.27μm (mean 5.49 μm; n = 50) and two to three apical appendages of 15.80-33.64μm (mean 25.87 μm; n = 50). These morphological characteristics are consistent with those of Neopestalotiopsis clavispora(Maharachchikumbura et al. 2012). The ITS (OM698813-15), tub2 (OM810165-67) andtef1α(OM810186-88) sequences were 99.38%, 99.09%, and 99.17% identical to the type N.clavispora strain MFLUCC12-0281 (accession nos. JX398979, JX399014, and JX399045) through BLAST analysis, respectively. A phylogenetic tree was generated using the concatenated sequences of ITS, tub2, and tef1α. The result showed that these three isolates were clustered with N.clavispora strains including the type MFLUCC12-0281. To perform pathogenicity tests, 20 healthy potted seedlings of A.corniculatum (2-year-old) were selected. Ten surface-sterilized leavesof 10 seedlings were wounded and inoculated by spraying conidial suspension ( 105conidia/ml). The same number wounded leaves of the other 10 plants treated with sterile water served as controls. All plants were wrapped in polyethylene bags for 24 h and incubated at 28°C in a growth chamber (at 90% relative humidity). After 10 days, all the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic. N. clavispora was reisolated from the lesions in terms of morphology and molecular characterization, whereas no fungus was isolated from the control leaves. The pathogenicity test was repeated three times under the same conditions. Thus, Koch's postulates were fulfilled. This pathogen has been reported on a wide host range worldwide, such as leaf spot on strawberry in China (Zhao et al. 2016) and twig blight on blueberry in Spain (Borrero et al. 2018). This is the first report of N. clavispora causing leaf blight on A.corniculatum in China.This study provides valuable information for the identification and control of the disease.

First report of Neopestalotiopsis clavispora causing leaf spot on Liquidambar formosana in China.

Liquidambar formosana Hance, a deciduous tree, is widely cultivated in China for its ornamental and afforestation value (Yin et al. 2021). In July 2019, leaf spot symptoms were observed with 20 to 30% disease incidence in Li shan forest farm (27°19'27.2″N, 115°32'51.08″E) in Ji'an city, Jiangxi province, China. Initial disease symptoms were small spots, which enlarged and circular to irregular, gray in the center, and dark brown to black circular on the lesion margin. Leaf pieces (5 × 5 mm) from the lesion borders were surfaced and sterilized in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water (Si et al. 2022). Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation, and the representative isolates, FX-2, FX-5, and FX-9 were used for morphological studies and phylogenetic analyses. The colonies of three isolates on PDA grew fast, covering the entire plate with white cottony mycelia with black acervuli after 8 to 10 days. Conidia were 5-celled, clavate to fusiform, smooth, 19.6-24.2 × 6.2-8.5 μm (n = 100). The 3 median cells were dark brown to olivaceous, central cell was darker than other 2 cells, and the basal and apical cells were hyaline. All conidia developed one basal appendage (3.5-8.2 μm long; n = 100), and 2-3 apical appendages (18-31 μm long; n = 100), filiform. Morphological features were similar to Neopestalotiopsis sp. (Maharachchikumbura et al. 2014). The internal transcribed spacer (ITS) regions, β-tubulin 2 (TUB2) and translation elongation factor 1-alpha (TEF1-α) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, T1/Bt-2b, EF1-728F/EF-2 (Maharachchikumbura et al. 2014), respectively. All sequences were deposited into GenBank (ITS, ON622512- ON622514; TUB2, ON676532 - ON676534; TEF1-α, ON676529 - ON676531). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed FX-2, FX-5, and FX-9 in the clade of N. clavispora. Based on the multi-locus phylogeny and morphology, three isolates were identified as N. clavispora. To confirm pathogenicity, 10 healthy 2-year-old seedlings, and 5 leaves per seedling were wounded with a sterile needle (Φ=0.5 mm) and inoculated with 200 μL conidial suspension per leaf(106 conidia/mL). Ten control plants were inoculated with ddH2O. All the inoculated leaves were covered with plastic bags and kept in a greenhouse at 26 ± 2 °C and RH 70%. All the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 8 days. N. clavispora was reisolated from the lesions, whereas no fungus was isolated from control leaves. N. clavispora can cuase leaf diseases in a variety of hosts, including × Taxodiomeria peizhongii (Zhang et al. 2022), Macadamia integrifolia (Qiu et al. 2020), Dendrobium officinale (Cao et al. 2022). N. cocoes, N. chrysea, Pestalotiopsis neglecta and P. neolitseae were also reported to infect L. formosana (Fan et al. 2021). However, this is the first report of N. clavispora infecting L. formosana in China. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

Fruit Rot on Persimmon Caused by Neopestalotiopsis saprophytica and Neopestalotiopsis ellipsospora in Guangxi, China.

Persimmon (Diospyros kaki Thunb.) is widely cultivated in China. On October 15, 2019, about 10% of persimmon fruits showed fruit rot in the orchards of Guilin, Guangxi, China (24°45' N, 110°24' E), which could cause more than 15% of yield losses. The initial symptoms of fruit rot exhibited irregular brown to black spots (range from 2 to 4 cm in diameter), the areas surrounding the blackened spots would be soft and rotten, and three diseased fruit samples were collected from three orchards, respectively. Tissues (5×5 mm) were cut from infected margins, surface-disinfected in 75% ethanol for 10 s, 2% NaClO for 2 min, rinsed three times in sterilized distilled water, and incubated on potato dextrose agar (PDA) at 25°C under 12/12 h light/darkness for a week. Forty-one tissues yielded morphologically similar cultures, and three representative isolates LPG1-1, LPG1-2, and YSG-1 were selected from three samples for further study, respectively. Their colonies showed wavy edges, white surfaces, and dense aerial hyphae on PDA after two weeks. Conidia were fusiform, straight to slightly curved, and 4-septate; basal cells were conical, hyaline, thin, and verruculose with two or three long and hyaline apical appendages and one short apical appendage; three median cells of LPG1-1 with length 14.06 to 17.69 μm (n=100), and LPG1-2 with length 14.03 to 17.61 μm (n=100) were dark brown to olivaceous, while three median cells of YSG-1 with length 12.54 to 15.58 μm (n=100) were dark brown. The conidial sizes of LPG1-1, LPG1-2, and YSG-1 were 17.41 to 27.68 × 4.63 to 8.55 μm (n=100), 18.06 to 27.41 × 4.33 to 8.21 μm (n=100), and 16.58 to 27.73 × 4.99 to 8.39 μm (n=100), respectively. The morphological characteristics were consistent with Neopestalotiopsis spp. (Maharachchikumbura et al. 2012; Maharachchikumbura et al. 2014). Primer pairs ITS4/ITS5, BT2a/BT2b, and EF1-526F/EF-1567R were used to amplify internal transcribed spacer (ITS), beta-tubulin (TUB2), and translation elongation factor 1 alpha (TEF1-α), respectively (Shu et al., 2020). All DNA fragments were sequenced bySangonBiotechCo., Ltd. (Shanghai, China). Sequences have been deposited in GenBank (ITS: OM349120 to OM349122, TUB2: OM688188 to OM688190, TEF1-α: OM688191 to OM688193). Based on BLASTn analysis of ITS, TUB2, and TEF1-α sequences, the LPG1-1 and LPG1-2 showed over 99% similarity to N. saprophytica, and YSG-1 showed over 99% similarity to N. ellipsospora. Phylogenetic analysis of the three isolates was performed with MEGA10 (version 10.0) based on sequences of ITS, TUB2, and TEF1-α using maximum parsimony analysis. The results revealed that LPG1-1 and LPG1-2 were clustered with N. saprophytica, and YSG-1 was clustered with N. ellipsospora. Pathogenicity tests of three isolates were conducted on 72 healthy persimmon fruits with and without wounds, and 9 fruits are for each treatment. The wound was made by a sterilized needle. Fruits were pre-processed with 75% ethanol for 10 s, 1% NaClO for 2 min and rinsed three times in sterile water. Conidial suspensions (10 µL, 106 conidia/mL in 0.1% sterile Tween 20) were inoculated on each site (4 sites/fruit). Control group was treated with 0.1% sterile Tween 20. All inoculated sites were covered with wet cotton. The inoculated fruits were placed in a plastic box to maintain humidity at 28℃. After 5 days, all wounded fruits showed fruit rot, whereas unwounded and control fruits remained asymptomatic, there were significant differences (P<0.05) in aggressiveness between N. saprophytica (average lesion diameter 13.1 mm) and N. ellipsospora (average lesion diameter 14.9 mm). Koch's postulates were fulfilled by re-isolating the causal agents from inoculated fruits. N. ellipsospora was previously reported as an endophyte in D. montana in southern India (Reddy et al. 2016). N. saprophytica could cause leaf spot of Erythropalum scandens and Magnolia sp., and fruit rot of Litsea rotundifolia in China and leaf spot of Elaeis guineensis in Malaysia (Yang et al. 2021, Ismail et al. 2017). To our knowledge, this is the first report of N. ellipsospora and N. saprophytica causing fruit rot on persimmon in the world. The results will provide a foundation for controlling fruit rot caused by pestalotioid fungi on persimmon.

First Report of Leaf Spot of Panax quinquefolius Caused by Pestalotiopsis nanjingensis in Tennessee and the United States.

American ginseng (Panax quinquefolius L.) is an herbaceous perennial understory plant. It was listed as endangered species by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (McGraw et al. 2013). Leaf spot symptoms were observed on 6-year-old cultivated American ginseng on a research plot (8 x 12 ft raised bed under a tree canopy) in Rutherford Co., TN in July 2021 (Fig. 1a). Symptomatic leaves were exhibiting light brown leaf spots with chlorotic haloes 0.5 to 0.8 cm in diameter, mostly confined within or bounded by veins. As the disease progressed, leaf spots expanded and coalesced into irregular shapes with necrotic centers, resulting in a tattered appearance of the leaf. Disease severity was about 50 to 80% of leaf area and incidence was 10% out of 20 plants. Plant tissues were surface sterilized with 10% NaOCl2 for 60s and washed thrice with sterile water and plated on potato dextrose agar (PDA). Colony growth of the isolates FBG880 and FBG881 on PDA were round, white, thick, and flocculent at the front of the plate and showed a yellowish-ringed shape on the back 10 days after incubation at 25°C (light/dark: 12/12h). Acervular conidiomata containing abundant conidia were observed on PDA. They were globose, 1.0 to 1.8 mm in diameter, and found as solitary or aggregated clusters. Conidia contained five cells (average 13.03±3.50 x 14.31±3.93 µm, n = 30). The middle three cells were light brown to brown. The basal and apical cells were nearly triangular, and transparent, with two to three (7:3 ratios, respectively) apical appendages (average 13.27±3.27 µm) and a basal appendage (average 4.50±0.95 µm, n = 30). To determine pathogen identity, total DNA was extracted using DNeasy PowerLyzer Microbial Kit from fungal colonies on PDA (isolates FBG880 and FBG881). The ribosomal internal transcribed spacer (ITS) region, beta-tubulin (BT), and translation elongation factors 1-α (EF1) genetic markers were amplified using ITS1/ITS4 (White et al. 1990), T1/T2 (Stefańczyk et al. 2016), and EF1/EF2 (O'Donnell et al. 1998), respectively. The sequences (GenBank accession nos. ITS: OQ102470 and OQ103415; BT: OQ107059 and OQ107061; and EF1: OQ107060 and OQ107062) are 100% similar to Pestalotiopsis nanjingensis (CSUFTCC16 and CFCC53882) (Jiang et al. 2022; Li et al. 2021) (Fig. 2). Based on morphology and molecular characteristics, the isolates were identified as P. nanjingensis. To conduct the pathogenicity trial, six healthy 1-year-old American ginseng plants, germinated from seeds and grown in the greenhouse were spray inoculated with a conidial suspension (1×106 conidia/ml) (FBG880). Six control plants were sprayed with sterile water. All plants were covered with plastic bags and incubated in a greenhouse set at 21 to 23°C, 70% relative humidity and 16-h photoperiod. After 48 h, bags were removed and plants were maintained under the same conditions. After one month, while control plants remained asymptomatic (Fig. 1b), inoculated plants started to exhibit symptoms resembling those in the research plot (Fig. 1c). Fungal isolates resembling P. nanjingensis in cultural characters were consistently recovered from inoculated plants and their identity as P. nanjingensis was confirmed by DNA sequencing. To our knowledge, this is the first report of leaf spot disease caused by P. nanjingensis on American ginseng. Identification of this pathogen and confirmation of its pathogenicity are fundamental to future disease management approaches.