Multigene Phylogenetic Analyses Research Articles

New Helminthosporium (Massarinaceae, Dothideomycetes) and Nigrospora (Incertae sedis, Sordariomycetes) species associated with walnut (Juglans regia L.) in China

Six collections of ascomycetes were obtained from samples collected from dead branches and leaves of Juglans regia in Guizhou and Yunnan provinces, China. By incorporating multigene phylogenetic analysis (ITS, LSU, rpb2, SSU, tef1-α, tub2) supplemented by morphological data, we establish two novel species, namely Helminthosporium guizhouense and Nigrospora yunnanensis. In morphology, H. guizhouense can be distinguished from H. caespitosum by its narrower conidia (13–16 µm vs. 27.3–35.5 µm), and N. yunnanensis is characterized by black, globose conidia (16.2 × 14.4 µm). The phylogenetic results further substantiated them as novel taxa. The present study contributes to our comprehension of the range of fungi found in Juglans regia, thereby expanding our knowledge of the diversity of fungi within this host.

Foliar blight, leaf spot, and stem blackening diseases caused by Stemphylium beticola, Alternaria alternata, and Alternaria atra on dragon's head (Lallemantia iberica) in Iran

Dragon's head, Lallemantia iberica, is an oilseed crop adapted to be grown in cold-dry areas. In the growing season of 2022–23 a severe foliar blight, leaf spot, and stem blacking symptoms were observed in commercial fields in northern and northwestern Iran, leading to more than 50 % yield loss. Infected samples were collected and transferred to the laboratory. Following the isolation of fungal agents, five representative isolates were selected for further studies. Morphological studies, based on conidial shape, conidiation, and other mycological characteristics, were carried out on a pure culture established from a single spore. Furthermore, multigene phylogenetic analyses were performed using sequences of translation elongation factor 1-α (TEF1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2), and Internal Transcribed Spacer (ITS) rDNA genes for Alternaria isolates, along with calmodulin (cmdA) for the Stemphylium isolate. The morphological and molecular studies identified the fungal isolates as Alternaria atra, Alternaria alternata, and Stemphyliium beticola. Pathogenicity tests on the commercial cultivar ‘Sara’ indicated that all isolates were pathogenic to dragon's head, but they differed significantly in aggressiveness. To the best of our knowledge, this is the first report of the association of these fungal species with an important foliar disease on this crop in Iran and worldwide.

Novel characterisation of Curvularia geniculata associated with leaf blight on Costus igneus in India

Costus igneus Nak, commonly referred as insulin plant or spiral flag, is a perennial herb belonging to the family Costaceae. Owing to its ability to regulate the blood sugar level, it is considered as a boon plant for diabetic patients, hence popularly known as the “sugar plant”. Although few foliar diseases have been documented on C. igneus, they generally occur in minor frequency under Indian conditions. However, in May 2022, a moderate to severe incidence of leaf blight was observed on insulin plants at University of Agricultural Sciences, GKVK, Bangalore, and nearby medicinal gardens. The affected leaves exhibited light brown lesions coalesced to form papery blight, gradually leading to complete drying of leaves. The current study aimed to identify, characterize, and document the fungal pathogen responsible for the disease, with a focus on its morphological, molecular, and biological characteristics to ensure accurate identification. Additionally, the study sought to determine optimal conditions for pathogen growth and sporulation to support the development of effective disease management strategies. The associated fungus was isolated, purified and identified as Curvularia geniculata through morphological and multigene phylogenetic analysis, using internal transcribed spacer (ITS) and large sub unit (LSU) regions of rDNA, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Biological characterization revealed that cornmeal agar (CMA) at 30°C and pH of 7 were ideal for better growth and abundant sporulation. These findings, highlighting the emergence of C. geniculata as a novel fungal pathogen infecting C. igneus in India, are crucial for developing effective disease management strategies to safeguard the medicinal properties of the plant.

Novel endophytic fungal species Pithoascus kurdistanensis producing morphine compounds

Papaver genus, commonly known as popies, is a valuable source of alkaloids used in medicine, including papaverine, morphine, codeine, and thebaine. We isolated six endophytic fungal isolates producing morphinan alkaloids from four Papaver species growing in Kurdistan Province, Iran. To do this, a 1:1 mixture of methanol and chloroform was used to extract fungal cultures. The contents of morphinan alkaloids in the extracts were subsequently determined using phase high-performance liquid chromatography. Among the morphinan alkaloid-producing fungal isolates, IRAN 4653C had the highest yield giving 23.06 (mg/g) morphine and 2.03 (mg/g) codeine when grown in potato dextrose liquid medium. The identity of this isolate was examined and recognized as a new fungal species named as Pithoascus kurdistanesis sp. nov. based on multi-gene phylogenetic analyses of ITS, TEF-1α, and TUB2 sequences data and morphological features. The morphinan-producing endophytic fungus and the isolated Pithoascus species from Papaver are being reported for the first time. Accordingly, this fungus shows promise as a new source of valuable compounds which is illustrated and introduced here as a new Microascaceae member belonging to Pithoascus from Kurdistan Province, Iran. Moreover, the morphinan productivity of P. kurdistanesis was further validated by gas chromatography-mass spectrometry (GC–MS).

Fusarium species associated with Euwallacea xanthopus in South Africa, including two novel species

ABSTRACT Ambrosia beetles (Coleoptera: Curculionidae: Scolytinae) are small wood-boring insects that live in an obligate symbiosis with fungi, which serve as their primary food source. Beetles residing in the genus Euwallacea have evolved a unique association with a clade of Fusarium that falls within the aptly named Ambrosia Fusarium Clade (AFC). The discovery of the invasive polyphagous shot hole borer, E. fornicatus, in South Africa, has heightened awareness of ambrosia beetles and their symbionts in the country. In this study, we investigated the Fusarium symbionts of three species of Euwallacea in South Africa, with a specific focus on those associated with E. xanthopus. Isolations of Fusarium strains from both living and dissected beetles yielded nearly 100 isolates. Using multigene phylogenetic analyses, these isolates were identified as six different Fusarium species. Fusarium hypothenemi and F. euwallaceae have previously been reported from South Africa. Fusarium pseudensiforme and Fusarium AF-6 are new records for the country. The remaining two species are new to science and are described here as F. rufum sp. nov. and F. floriferum sp. nov. Targeted fungal isolation from specific beetle body parts revealed that the AFC species collected were typically associated with the dissected beetle heads and helped us identify the likely nutritional symbiont of E. xanthopus. This study highlights the understudied diversity of fungal associates of ambrosia beetles present in South Africa.

First Report of Alternaria alternata Causing Leaf Spot of Magnolia delavayi in China

Magnolia delavayi, known as Delavay's magnolia or Chinese evergreen magnolia, is an enchanting deciduous tree native to southwestern China (Xu et al. 2020). In March 2024, leaf spots were observed on M. delavayi plants at Qujing Normal University (25.527°N, 103.744°E), Qujing, Yunnan, China. About 80% of the trees (n=92) on campus were infected, with infected leaves accounting for about 10 to 30% of all leaves. The leaf spots exhibited a round or oval shape, with a yellowish-brown center and a brown outer ring. As the spots expanded, the leaves gradually dried up and fell off. Five symptomatic leaves were collected from the middle sections of five diseased plants. Tissue samples (5 × 5 mm) were excised from the lesion margins and subjected to surface sterilization in 75% ethanol for 30 seconds and 1% sodium hypochlorite for 60 seconds. They were then rinsed three times with sterile water and dried on sterilized paper. The samples were plated on potato dextrose agar (PDA) and incubated at 28 °C for 3 days. Developing colonies were retransferred to new PDA and purified by the hyphal tip technique (Senanayake et al. 2020). Two representative isolates (SYL1 and SYL2) were selected and confirmed to be the same species based on morphological characteristics and molecular identification. On PDA, the fungal colonies initially appeared grayish-white and turned dark gray over time with cotton-like aerial hyphae on the surface. The conidia are light brown, pear-shaped or rod-shaped, with 1 to 4 transverse septa and 0 to 1 oblique septa, and measured 5.4 to 12.7 × 10.4 to 25.7 μm (n = 50). Molecular identification was performed by partial sequencing of the internal transcribed spacer (ITS: ITS4/ITS5) region, glyceraldehyde 3-phosphate dehydrogenase (GAPDH:gpd1/gpd2), translation elongation factor 1-alpha (TEF1:EF1/EF2), and Alternaria major allergen (Alt a 1:Alt-for/Alt-rev) (Woudenberg et al. 2015). The resulting sequences of ITS (PP951882, PP951883), GAPDH (PP968942, PP968943), Alt a 1(PP968940, PP968941) and TEF1 (PP968938, PP968939) were deposited in GenBank. BLAST analyses revealed that the sequences from these two isolates showed 100% identity with those from Alternaria alternata strains for each gene. Additionally, a multigene phylogenetic analysis revealed a distinct group containing SYL1, SYL2 and other A. alternata strains with a posterior probability of 100%. The morphological and phylogenetic evidence collectively suggested that the two isolates belonged to A. alternata. Strain SYL1 was used for the pathogenicity test on three 5-year-old M. delavayi plants on campus. Three healthy leaves of each plant were punctured with a sterile needle and sprayed with 20 μl of spore suspension (1 × 106 conidia/ml). Similarly, another nine wounded leaves were sprayed with 20 μl of sterile water. All inoculated leaves were enclosed in plastic bags for 48 hours. Disease symptoms were observed on leaves inoculated with spore suspension after 7 days, while control plants remained healthy. This experiment was repeated three times with same results. To fulfill Koch’s postulates, A. alternata was reisolated from the inoculated leaves and identified based on morphological characteristics and ITS sequencing. To our knowledge, this is the first report of A. alternata causing leaf spot on M. delavayi in China. This research expands the known host range of this pathogen and provides relevant information to the design of disease management strategies.

Insights from multigene analysis: first report of a Southeast Asian Mosquito, Aedes (Mucidus) laniger (Diptera: Culicidae) on Jeju Island from Korea

BackgroundCertain mosquitoes are known as dominant vectors worldwide, and transmit infectious diseases. The expansion of mosquito habitats due to climate change and increased human activities poses a significant health threat by facilitating the spread of various non-native infectious diseases. This study focused on the detection of the Southeast Asian mosquito species, Aedes (Mucidus) laniger (Wiedemann, 1820) on Jeju Island, the southernmost region of the Republic of Korea (ROK), highlighting the potential risks associated with the spread of vector-borne diseases, particularly emphasizing the elevated likelihood of invasion by Southeast Asian mosquitoes.MethodsField surveys were conducted in August 2023 on Jeju Island. Adult mosquitoes were collected using BG-sentinel traps and identified to the species level using taxonomic keys. Morphological and molecular analyses were employed to confirm species designations. Molecular data, including mitochondrial and nuclear genes, were used for phylogenetic analysis, which was performed to compare and identify among recorded subgenera in ROK. Species distribution modeling for Ae. laniger was performed to predict potential habitats using R package ‘BIOMOD2’.ResultsThe two specimens of Ae. laniger were collected for the first time on Jeju Island. Morphological and molecular analyses confirmed the identity of this species within the subgenus Mucidus and validated the first record of this species in the ROK. We employed a simple multigene phylogenetic analysis to confirm a new mosquito record at the genus and subgenus levels, finally validating the consistency between morphological identification and molecular phylogenetic outcomes. Furthermore, we have updated the taxonomic keys for the genus Aedes in the ROK, and revised mosquito lists for Jeju Island, incorporating the inclusion of Ae. laniger. On the basis of species distribution modeling, the area of suitable habitat for Ae. laniger is expected to expand due to climate change, but this change did not appear to be meaningful in East Asia.ConclusionsThis case offers the first report of the Southeast Asian mosquito, Ae. laniger, in the ROK. The detection of this species on Jeju Island suggests the potential establishment of a breeding population their habitat and raises concerns about further expansion into the Korean Peninsula. Considering the annual occurrence of mosquito-borne disease cases in the Southeast Asia, it is essential to conduct monitoring not only in Jeju Island, where Ae. laniger has been identified, but also across the entire Korean Peninsula.Graphical

Multi-Gene Phylogenetic Analyses Reveals Heteroxylaria Gen. Nov. and New Contributions to Xylariaceae (Ascomycota) from China.

An in-depth study of the phylogenetic relationships of Xylaria species associated with nutshells of fruits and seeds within the genus Xylaria and related genera of Xylaceaecea was conducted in China. The multi-gene phylogenetic analyses were carried out based on ITS, RPB2, and TUB sequences of 100 species of 16 known genera in Xylariaceae around the world. Based on molecular phylogenetic analyses, morphological observations, and ecological habitats, a new genus, Heteroxylaria, is established to accommodate four new species, viz. H. cordiicola, H. juglandicola, H. meliicola, and H. terminaliicola, and four new combinations, viz. H. oxyacanthae, H. palmicola, H. reevesiae, and H. rohrensis. The genus is characterized by cylindrical stromata with conspicuous to inconspicuous perithecial mounds, surface black, having brown to dark brown ascospores with a germ slit, and it grows on nutshell of fruits. The combined ITS+RPB2+TUB sequence dataset of representative taxa in the Xylariaceae demonstrate that Heteroxylaria is grouped with Hypocreodendron but forms a monophyletic lineage. All novelties described herein are morphologically illustrated and compared to similar species and phylogeny is investigated to establish new genera and species.

Broadening the horizon of Indian mycobiota: two new species and one new generic record in Boletoid mushrooms (Boletaceae)

A combination of morphological studies and multigene molecular phylogenetic analyses unveiled two significant novel species of boletes namely Anthracoporus indohimalayanus sp. nov. and Boletus dhakuricus sp. nov. from the states of Sikkim and Uttarakhand, respectively. The first novel species, i.e. A. indohimalayanus is also a new generic record of the genus Anthracoporus from India, whereas the second novel species i.e. B. dhakuricus is a member of wild edible porcini mushroom. Both the taxa are described with their morphological details and molecular phylogenetic inferences.

Two new species of Nectriaceae (Hypocreales, Sordariomycetes) from Yunnan, China.

Nectriaceae is a highly diverse family, and members have a worldwide distribution, particularly in warm temperate to tropical regions. During the survey of fungal diversity in different habitats in Yunnan province, China, two new species isolated from soil and air respectively, namely Atractiumyunnanense sp. nov. and Nalanthamalaxishuangbannaensis sp. nov., were proposed based on morphological comparisons and the multi-gene phylogenetic analyses of combined ITS, LSU, rpb2, and tub2 sequence data. Phylogenetically, both species clustered in a monophyletic clade within Nectriaceae with strong support. A.yunnanense is characterized by synnematous conidiophores, pale olivaceous-green, clavate to oblong-ellipsoidal, multi-septate conidia, and pale olivaceous-green chlamydospores. N.xishuangbannaensis has acremonium-like or penicillium-like conidiophores and either obovate or ellipsoidal, cylindrical or fusiform conidia. Full descriptions, illustrations, and a phylogenetic tree showing the phylogenetic position of the two new species were provided.

Three new species of Colletotrichum (Glomerellales, Glomerellaceae) associated with walnut (Juglansregia) anthracnose from China.

Colletotrichum species are significant pathogens of various economic plant hosts worldwide. In this study, 45 Colletotrichum isolates were obtained from symptomatic walnut leaves of walnut anthracnose in Shaanxi and Sichuan Provinces. In conjunction with morphological evidence and multi-gene phylogenetic analyses of internal transcribed spacer (ITS), actin (act), chitin synthase 1 (chs1), glyceraldehyde-3-phosphate dehydrogenase (gapdh) and beta-tubulin (tub2) sequences support the introduction of three new species, namely Colletotrichumcordae, C.guangyuanense and C.juglandium. Five species of Colletotrichum were identified to be C.fioriniae of the C.acutatum species complex, C.karsti of the C.boninense species complex, C.gloeosporioides, C.mengyinense and C.siamense of the C.gloeosporioides species complex. The three new species are described and illustrated in this paper and compared with taxa in the Colletotrichumgloeosporioides species complex. The current results improve the understanding of Colletotrichum species causing walnut anthracnose in China.

Morpho-molecular characterization of phoma-like fungi from Morus alba in northern Thailand; a novel species (Boeremia albae) and a new host record (B. maritima)

Boeremia was established to accommodate phoma-resembling fungi. Its species occur in terrestrial ecosystems as endophytes, saprobes and pathogens, except one species reported from a marine ecosystem. Boeremia species are characterized by hyaline, thin-walled, and aseptate (occasionally 1(–2)-septate) conidia that are variable in shape, and hyaline, straight or slightly curved, thick-walled, and 1-septate ascospores that are usually constricted at the septum. In the past, host associations were used to delimit Boeremia species. However, since Boeremia taxa have overlapping morphological characters and are cryptic, it renders taxonomic identification arduous. Therefore, the use of other approaches including multi-gene phylogenetic analyses are imperative. Recommended DNA markers for species delineation are the internal transcribed spacer (ITS, nuclear rDNA consisting of ITS1-5.8S-ITS2) and large subunit (28S, D1–D2 domains of nuclear 28S rDNA) loci, and the genes for actin (ACT1), beta-tubulin (TBB1), RNA polymerase 2 (RPB2) and translation elongation factor 1α (TEF1). Here, we applied morphological and molecular phylogenetic analyses to establish a new taxon (B. albae), and a new host and geographical record for B. maritima associated with leaf spots of Morus alba (Moraceae) in northern Thailand. By providing sequence data for three additional gene regions, our phylogenetic analyses impart a stable phylogenetic placement of the ex-type strain of B. maritima, as illustrated. This is the first study that reports Boeremia species from M. alba, and B. maritima from a terrestrial habitat.

First report of Morchella galilaea, an autumn species from Pakistan, based on multilocus DNA sequences, light microscopy, and scanning electron microscopy analyses.

Morchella is highly prized for its distinct and delicious taste, making it one of the most sought-after and valuable macrofungi. Despite Pakistan exporting morels to various European countries, Morchella's diversity in this South Asian country has not been cataloged using multilocus DNA sequence data. Realizing the need for their scientific identity, various collections were commissioned, including those from the low and high altitudinal zones of the Charsadda, Swat, and Poonch districts of Pakistan during autumn of 2015 through 2019. Specimens were identified by sequencing the internal transcribed spacer (ITS) region and portions of the RNA polymerase II largest subunit (RPB1), second largest subunit (RPB2), and translation elongation factor 1-α (TEF1) loci. Multigene molecular phylogenetic analysis revealed the autumnal collections were Morchella galilaea Masaphy & Clowez. The species was previously documented in the United States, China, Israel, and Turkey. A detailed taxonomic description, light and scanning electron microscopy, and multigene molecular phylogenetic analyses of the species are reported here for the first time from Pakistan. The study underscores the necessity for further surveys of unexplored areas of Pakistan and systematics of Morchella to augment species diversity and their geographic distribution. RESEARCH HIGHLIGHTS: Specimens of Morchella were collected from low and high altitudinal zones in Pakistan during the autumn. Specimens were assessed using multigene (ITS, RPB1, RPB2, and TEF1) molecular phylogenetic analyses for the first time in Pakistan. The specimens were identified as Morchella galilaea based on multigene molecular phylogenetic analysis. Updated taxonomic descriptions of macro- and microanatomical characters were provided for clarity and precision. Hitherto-unreported shapes of paraphyses and acroparaphyses are observed and thoroughly reported. Scanning electron microscopy (SEM) of an ascospore is provided.

Morphological and Phylogenetic Analyses Reveal Three Novel Species of Sanguinoderma (Ganodermataceae, Basidiomycota) from Yunnan Province, China.

Sanguinoderma (Ganodermataceae) is recognized as a valuable medicinal resource in Taiwan, China. Additionally, it serves as a traditional folk medicine for treating neurotic epilepsy in Malaysia. This study involved the collection of six specimens of Sanguinoderma from Yunnan Province, China. Employing multigene phylogenetic analysis of DNA sequences, including internal transcribed spacer (ITS), nuclear large subunit (LSU), RNA polymerase II second largest subunit (rpb2), translation elongation factor 1-alpha (tef1-α), mitochondrial small subunit (mtSSU), nuclear small subunit (nSSU) and morphological examinations, three new species, viz. Sanguinoderma concentricum, S. dehongense and S. ovisporum, are introduced. Sanguinoderma concentricum is characterized by a central stipe basidiomata, an orbicular to suborbicular pileus, a grayish-yellow surface with alternating concentric zones and wavy margin-like petals and regular pileipellis cells (4-8 × 17-28 μm). Sanguinoderma dehongense is characterized by a long stipe and flabelliform basidiomata, a dark-grayish yellow-to-dark-yellow pileus surface, irregular pileipellis cells and wavy margin and ellipsoid basidia (8-11 × 9-13 μm). Sanguinoderma ovisporum is characterized by a reniform basidiomata, a heterogeneous context and ovoid basidiospores (7.5-8.6 × 5.5-7.2 µm). A detailed description and illustrations of these new species are provided, as well as a morphological comparison with similar taxa.

Four new species of Candolleomyces (Psathyrellaceae) from the Punjab Plains, Pakistan

ABSTRACT The purpose of this study is to explore the genus Candolleomyces from Pakistan. Fungal surveys were conducted in different sites of the Punjab plains during the 2016–2022 period. Several specimens belonging to the family Psathyrellaceae were collected. Traditional morphology and multigene phylogenetic analyses confirmed the novelty and placement of the species in genus Candolleomyces. The phylogenetic study of the nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 (ITS) and partial 28S rRNA regions confirmed the distinct positions of four new taxa, Candolleomyces iqbalii, C. kotadduensis, C. pakistanicus, and C. parvipileus, in the genus. Currently, the genus consists of 43 species worldwide. However, with the inclusion of these four species, the total number has risen to 47. Detailed descriptions, phylogeny, comparisons, and illustrations are provided.

First Report of Rosa roxburghii Rot Disease Caused by Fusarium annulatum in China.

Rosa roxburghii Tratt is a rosaceous shrub originating from southwest China (Fu et al. 2020). From September to October 2022, R. roxburghii rot occurred in Guizhou Province, China, within a major R. roxburghii production area covering from 5 ha to 50 ha, with an incidence rate of 10 to 15%. Symptoms manifested as black and brown lesions on the fruit surface, which were concave, soft, foul-smelling, and surrounded by grayish-brown tissue. Three infected R. roxburghii shrubs were randomly collected from each household, placed in transparent plastic bags, and pathogen isolation was conducted in a laboratory. Infected R. roxburghii fruits were surface-sterilized with 0.5% NaOCl for 2 min, rinsed five times with sterile water, and dried. Symptomatic tissues from the margin between necrotic and healthy tissues were cut into 5 × 5 mm pieces, placed onto potato dextrose agar (PDA), and incubated at 28ºC in the dark for 5 days. Hyphal tips of fungi growing from the samples were transferred onto new PDA plates and incubated until they produced conidia. A total of five fungal isolates with similar morphological characteristics were obtained. The colony obtained by single-spore purification was light purple to dark purple with abundant aerial mycelium. Macroconidia were relatively slender with a curve and zero to three septate. Microconidia appeared obovoid to pyriform, with sizes of 5.2 to 17.2 × 2.1 to 3.3 μm (n = 50). The morphological characteristics were consistent with Fusarium annulatum (Yilmaz et al. 2021). Genomic DNA of two representative isolates (Zhaochanglin 1621 and 1622) was extracted using the DN14 cetyltrimethylammonium bromide rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd, Beijing). The TEF1 and RPB2 gene were amplified by Polymerase Chain Reaction using primers EF1-983F/EF1-2218R (Rehner et al, 2005), bRPB2-6F/bRPB2-7.1R (Matheny et al, 2002), respectively. All sequences were deposited in GenBank (TEF1, PP236860, PP236861; RPB2, PP767864, PP767865). BLAST searches of the TEF1, and RPB2 sequences revealed the TEF1 sequences had 99.89% (937/938 bp) identity with F. annulatum isolate CBS 258.54; and the RPB2 sequences had 99.86% (737/738 bp) identity to isolate CBS 267.93. In the phylogenetic tree, the isolates (Zhaochanglin 1621 and 1622) clustered with the representative strains of F. annulatum. The morphology and multi-gene phylogenetic analysis indicated that is the isolates were F. annulatum. To complete Koch's postulates, five mature, healthy R. roxburghii fruits were surface disinfected with 1% NaClO solution for 1 min, rinsed with sterile water, and dried at 25℃ for 30 min. A conidial suspension (106 spores/ml) collected from two isolates (Zhaochanglin 1621 and 1622) was sprayed onto R. roxburghii fruits, and the control treatments were sprayed with sterile distilled water. All R. roxburghii fruits were incubated at 25 ºC with 80% relative humidity. The experiment had five replicates. After 7 days of incubation, all the inoculated fruits showed similar symptoms to those initially observed on the originally infected plants. The same pathogen was reisolated and identified by morphological character ization and molecular analysis, fulfilling Koch's postulates. Thus, the pathogen causing rot of R. roxburghii was determined to be F. annulatum (H. Zhang et al, 2024). To our knowledge, this is the first report of F. annulatum causing R. roxburghii rot disease in China. F.annulatum has a wide range of hosts and has been reported to infect a wide range of crops, fruits, and vegetables (Bacon and Nelson 1994). This study lays a foundation for further study and developing disease control methods and the improvement of the economic benefits of R. roxburghii.

First Report of Neofusicoccum yunnanense Causing Leaf Spot on Ivy in Yunnan Province, China.

Ivy (Hedera nepalensis var. sinensis (Tobl.) Rehd) is an evergreen root-climbing vine, widely cultivated in eastern Asia because of its ornamental, environmental, and medicinal value (Wu et al. 2019). In October 2023, the leaf spot symptom of Ivy was observed in the Kunming Botanical Garden in Yunnan Province, China (25.14°N, 102.75°E), and the disease incidence was up to 38% (76 of 200 leaves). Initially, dark-brown or black small spots appeared on the leaves. As the lesions progressed, their center emerged tawny, and the black halos were expanded around the lesions. In severe cases, small spots could link together to form leaf blight even resulting in blade death. In order to obtain pure isolates, 10 diseased leaves were collected and cut into 1 mm × 1 mm pieces, surface disinfected with 75 % ethanol for 30 s, followed by 3% NaClO for 3 min, and finally washed three times with sterilized water. The pieces were placed on the potato dextrose agar (PDA) media, which was incubated at 25°C for 3 days. Individual hyphal tips from the developing fungal colonies were placed on PDA and incubated for 5 to 10 days. Six strains (6 out of 10) were obtained with same colony morphology. Conidia were hyaline, unicellular, nonseptate, ellipsoidal or fusiform, thin walled, externally smooth and ranged from 15.0 to 22.0 (avg. 18.4) μm × 5.0 to 8.0 (avg. 7.2) μm (n=30). Morphological comparison proposed that the present fungi belonged to Neofusicoccum (Zhang et al. 2021). Two isolates (GUCC23-0141 and GUCC23-0142) were selected for multi-gene phylogenetic analyses. The PCR reaction of the internal transcribed spacer region (ITS), translation elongation factor 1-α (EF1-α), and β-tubulin genes were run using primers ITS1/ ITS4 (White et al. 1990), EF1-728F/ EF1-986R (Carbone and Kohn 1999), and Bt2a/ Bt2b (Glass and Donaldson 1995). The accession numbers of DNA sequences of GUCC23-0141 and GUCC23-0142 are ITS: PP728109 and PP728110; TUB2: PP744490 and PP744491; and TEF1-α: PP744488 and PP744489. BLAST analysis showed 100% identity for ITS and TUB2, and 98.97% for TEF1-α with the Neofusicoccum yunnanense (CSF6142). Phylogenetic analyses also supported that our isolates kept a close relationship to N. yunnanense. Three one-year plants were used for pathogenicity test, two of which were inoculated with PDA plugs containing N. yunnanense and one of which was inoculated with blank PDA plugs and used as control. For each plant, three leaves were selected to conduct the test, whose surfaces were sterilized with 75% alcohol. All the leaves were covered with cotton moistened with sterilized water on top. All plants were placed in a greenhouse with 25℃ and 75% humidity. Few small black spots were observed at the inoculation site after 3 days, which were enlarged gradually after 7 days. However, control plants remained healthy. N. yunnanense was reisolated from the diseased tissues and identified based on morphological and molecular characteristics. On basis of pathogen identification and Koch's test, we proposed the leaf spot of Ivy caused by N. yunnanense. This was the first report about N. yunnanense causing the disease of Ivy. This result provides a theoretical basis for further research into the control of the disease. As an important ornamental plant, we should pay attention to the management of this disease.

Thyridium livistonae sp. nov. from Yunnan Province, China, with two new combinations

ABSTRACT An ascomycetous fungus was collected and isolated from dead leaves of Livistona chinensis in Lincang City, Yunnan Province, China, and morphology and multi-gene phylogenetic analyses of LSU, ITS, Actin, and β-tub were carried out to identify it. The morphology and multigene phylogeny revealed that this fungus is a distinct species of Thyridium. Therefore, this fungus is described and introduced as Thyridium livistonae sp. nov. In addition, two new combinations T. hipposidericola and T. xishuangbannaense are proposed based on morphology and phylogeny for Phialemoniopsis hipposidericola Karun., Tibpromma & X.F. Liu and P. xishuangbannaensis Karun., Tibpromma & X.F. Liu respectively. A detailed description, illustrations, and phylogenetic results are provided for the new species and two new combinations.

Two New Species of the Genus Diderma (Physarales, Didymiaceae) in China with an Addition to the Distribution.

Myxomycetes are an important component of terrestrial ecosystems, and in order to understand their diversity and phylogenetic relationships, taxonomic issues need to be addressed. In our 1985-2021 biodiversity investigations in Shaanxi Province, Jilin Province, the Inner Mongolia Autonomous Region, Hubei Province, and Henan Province, China, Diderma samples were observed on rotten leaves, rotten branches, and dead wood. The samples were studied, based on morphological features coupled with multigene phylogenetic analyses of nSSU, EF-1α, and COI sequence data, which revealed two new species (Diderma shaanxiense sp. nov. and D. clavatocolumellum sp. nov.) and two known species (D. radiatum and D. globosum). In addition, D. radiatum and D. globosum were newly recorded in Henan Province and the Inner Mongolia Autonomous Region, respectively. The paper includes comprehensive descriptions, detailed micrographs, and the outcomes of phylogenetic analyses for both the newly discovered and known species. Additionally, it offers morpho-logical comparisons between the new species and similar ones.

Outbreak of Alternaria Black Spot of Pomegranate (Punica granatum L.) in Italy as a Consequence of Unusual Climatic Conditions.

Alternaria black spot of pomegranate (Punica granatum) was reported for the first time in Italy. In spring 2023, an outbreak of this disease was noticed in commercial pomegranate 'Wonderful' orchards of the municipality of Misterbianco (Sicily), following an unusually rainy period. A total of 30 randomly selected Alternaria isolates recovered from typical necrotic spots of leaves and fruits were characterized. Based on the colony morphology on solid agar media (PDA and MEA), isolates were separated into three distinct morphotypes (1, 2, and 3). The first two morphotypes comprised only isolates from fruits, while morphotype 3 comprised only isolates from leaves. Multigene phylogenetic analysis of four DNA regions, including internal transcribed spacer (ITS), translation elongation factor 1-α (EF-1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and a SCAR marker (OPA10-2), identified the isolates of morphotypes 1 and 2 as Alternaria alternata and morphotype 3 isolates as A. arborescens. In pathogenicity tests on unwounded leaves and fruit, the isolates of all three morphotypes produced symptoms on the leaves of three pomegranate cultivars, 'Acco', 'Wonderful', and 'Etna'. The symptoms on 'Acco' leaves were the least severe. Conversely, the fruits of 'Acco' were the most susceptible. The isolates of morphotypes 2 and 3 were not pathogenic on the fruits of 'Wonderful' and 'Etna'. This is the first report of Alternaria black spot in Italy and of A. arborescens associated with Alternaria black spot of pomegranate worldwide.