Morphological and Molecular Characterization of Botryosphaeria wangensis Causing Branch Blight of Acer saccharum in China

Mycoleptodiscus terrestris (Gerd.) Ostaz. has been studied extensively as a potential mycoherbicide against aquatic weeds since the early 1970s (Mathur and Gehlot 2018; Shearer 1998). However, it is also a pathogen on many legumes including alfalfa (Gerdemann 1953; Smith et al. 1998). In August 2019, alfalfa plants with stunted yellow foliage and rotted stems at the base of the crown were observed in a variety trial planted in spring 2018 at the University of Minnesota, St. Paul, Minnesota. Affected plants were in patches across the field with approximately 20% of plants showing symptoms. Plants from patches had few lateral and fibrous roots and dark lesions appeared in the crown and root tissue. Tissue samples (5 mm2) from 10 plants with symptomatic crown and root tissues were surface disinfested with 70% ethanol for 5 min, followed by 30 s in 10% NaOCl, and then rinsed three times in sterile distilled water. Samples were air-dried and placed on water agar (WA) amended with 25 µg/ml rifampicin. Plates were incubated at room temperature for 3 days and fungal hyphal tips from tissue samples were transferred to potato dextrose agar (PDA). After 7 days, isolates were tentatively identified by morphological characteristics as M. terrestris (Ostazeski 1967). The hyaline mycelia turned from olive-gray to dark gray with age. Abundant dark microsclerotia formed 5 days after incubation that varied in size and shape, measuring 300-860 x 270-600 µm. Molecular identification of two representative isolates (DAZ5 and DAZ9) was done by sequencing the internal transcribed spacer (ITS) regions of the rDNA, the translation elongation factor 1α (TEF1), and the second largest subunit of RNA polymerase II (RPB2) genes. For both isolates the ITS, TEF1, and RPB2 genes were amplified and sequenced with primers ITS1/4 (White et al. 1990), 983F/1567R (Rehner and Buckley 2005), and fRPB2-7cR/RPB-5F2 (Liu et al. 1999; Sung et al. 2007), respectively. ITS amplification conditions followed White et al. (1990), the TEF and the partial RPB2 gene was obtained by using a touchdown PCR protocol as described in Rehner and Buckley (2005) and Woudenberg et al. (2017), respectively. Based on a BLAST search of the NCBI nucleotide database, the ITS sequences (GenBank MN851265.1 and MN851266.1) had 100% identity with M. terrestris strain CBS 231.53 (MK487754.1). The TEF1 (MN873019, MN873020) and RPB2 sequences (MN873021, MN873022) had 100% identity with M. terrestris strain IMI 159038 (MK495977.1, MK492735.1). A pathogenicity test was performed by inoculating five 3-week-old alfalfa plants per cultivar (cv. DKA44-16RR, Vernal, 53V52, and Agate) with isolates DAZ5 and DAZ9. Plants were inoculated around the exposed stem base with three 5 mm diameter PDA plugs from a culture of M. terrestris covered with microsclerotia. Control plants were inoculated with sterile PDA plugs. After inoculation plants were incubated at 25 0C with a 16-h photoperiod in a growth chamber. The first symptoms appeared two months after inoculation on all cultivars as a dark lesion at the stem base followed by yellowing and rotting of stems. Inoculated plants had more fibrous roots than the controls. The control plants were symptomless with healthy root development. The pathogen was re-isolated from all infected alfalfa cultivars and had the same morphology as isolates DAZ5 and DAZ9, fulfilling Koch's postulates. To our knowledge, this is the first report of M. terrestris causing crown and root rot disease of alfalfa in Minnesota.

ABSTRACTIn the spring of 2023 in Eboli and Caserta (Campania, southern Italy), strawberry plants (var. Marimbella) grown in organic open fields showed an outbreak of a severe and unprecedented decline (disease incidence reaching > 80%) associated with root rot, crown rot, and leaf spot and closely resembling symptoms reported previously in other countries for Neopestalotiopsis spp. infection. Therefore, the present study was undertaken with the aim of determining the aetiology of this serious disease. Fungal isolates were obtained from symptomatic strawberry plants and investigated in detail for molecular identification. Phylogenetic analysis was conducted by amplifying and sequencing three DNA barcodes: the internal transcribed spacer (ITS) region of rDNA, the β‐tubulin (tub2) partial gene, and the translation elongation factor 1α (tef1) partial gene. Symptoms observed in the field were replicated in pathogenicity tests, conducted by inoculating strawberry (var. Marimbella) leaves, fruits and plants, thus satisfying Koch's postulates. Phylogenetic analyses identified the causal agent as Neopestalotiopsis rosae. To our knowledge, this is the first report of the emerging and serious fungal pathogen N. rosae infecting strawberry in Italy.

Selenicereus megalanthus (family Cactaceae), commonly known as yellow pitahaya is a new crop being planted commercially in Malaysia. In May 2021, stem canker symptoms with sign of black pycnidia formed on the surface of canker (30- to 55-mm in diameter) were observed on the stem of 80% of 'yellow pitahaya' plants in the field (~8 ha) located in the district Keningau of Sabah, Malaysia (5°20'53.1"N 116°06'23.0"E). The infected stems became rotted when black pycnidia formed. To isolate the pathogen, the symptom margin was excised into four small blocks (5 x 5 x 5 mm), and the blocks were surface sterilized based on Khoo et al. (2022) before plating on potato dextrose agar (PDA). Plates were incubated at 25°C for 7 days in the dark. Two isolates were obtained and were named Keningau and Keningau02. Powdery white mycelia were initially observed in two plates, and then became dark grey with age. Dark pigmentation in plates was observed after a week of incubation at 25°C in the dark. Arthroconidia (n= 30) were hyaline to dark brown, circular or cylindrical with round to truncate ends, with zero to one septum, measuring 8.9 x 5.6 µm in size. Conidia (n= 30) exuded in milky white cirrhus from pycnidia were one-celled, aseptate, oblong, measuring 10.3 × 4.6 µm in size. When reached the maturity stage, conidia were brown and septate. Genomic DNA from Keningau and Keningau02 were extracted from fresh mycelia based on Khoo et al. (2021) and Khoo et al. (2022). Amplification of the internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-α (TEF1) region and β-tubulin (TUB) genes were performed using ITS1/ITS4, EF1-728F/EF1-986R and T10/Bt2b primer sets, respectively (Carbone and Kohn, 1999; O'Donnell et al. 1997; White et al. 1990). The products were sent to Apical Scientific Sdn. Bhd. for sequencing. BLASTn analysis of the newly generated ITS (GenBank OK458559, OM649909), TEF1 (GenBank OM677768, OM677769) and TUB (GenBank OL697398, OM677766) indicated 99% identity to Neoscytalidium novaehollandiae strain CBS 122071 (GenBank MT592760). Phylogenetic analysis using maximum likelihood and Bayesian inference on the concatenated ITS-TEF1-TUB was constructed using IQ-Tree and MrBayes3.2.7. Neoscytalidium hyalinum, N. novaehollandiae and Neoscytalidium orchidacearum are reduced to synonymy with N. dimidiatum (Philips et al. 2013; Zhang et al. 2021). Although N. novaehollandiae is morphologically and phylogenetically similar to N. dimidiatum, but N. novaehollandiae produce muriform, Dichomera-like conidia that distinguish this species from other known Neoscytalidium species (Crous et al. 2006). No muriform, Dichomera-like conidia were observed in the Malaysia' isolates. The pathogen was identified as N. dimidiatum based on molecular data and morphological characterization (Serrato-Diaz and Goenaga, 2021). Pathogenicity tests were performed based on Mohd et al. (2013) by injection inoculation of 0.2 ml of conidial suspensions (1 x 106 conidia/ml) from isolate Keningau to three 30-month-old yellow pitahaya stems using a disposable needle and syringe. Distilled water was injected into three mock controls. The inoculated yellow pitahaya plants were covered with plastics for 48 h and incubated at 25°C. The pathogenicity test was also performed using isolate Keningau02. All inoculated stems developed symptoms as described after 6 days post-inoculation, whereas no symptoms occurred on controls, thus fulfilling Koch's postulates. The experiments were repeated two more times. The reisolated fungi were identical to the pathogen morphologically and molecularly. To our knowledge, this is the first report of N. dimidiatum causing stem canker on S. megalanthus in Malaysia. Our findings serve as a warning for the authorities and farmers that the disease threat has appeared in the Malaysian yellow pitahaya production.

Eucalypt species are among the most important for timber production worldwide. Eucalyptus cloeziana is increasingly culticated due to its desirable structural properties. Leaf blight is one of the most devastating diseases of E. cloeziana in China. In May 2019, leaf blight samples were collected from E. cloeziana in Chongzuo, Guangxi, China (22°20'37.70"N, 107°49'29.29"E). Lesions began at the leaf margin and extended to 1/4-3/4 of the total leaf surface area. Lesions (26.76±12.64 mm diameter) were round, yellow, and withered in appearance, and sometimes many black, round pycnidia were observed. Leaves with blight were collected randomly from 10 E. cloeziana plants. Tissue blocks (3 mm×3 mm) were sampled from diseased and healthy leaf portions, then surface disinfected with 75% ethanol for 20 s and 0.1% HgCl2 for 3 min. After washing with sterile water three times, dry tissue blocks were placed on potato dextrose agar (PDA) medium and incubated at 28°C for 5 days. Hyphae were milky white or whitish, and sparse. The colonies had petal-shaped edges and the conidiophores were clustered, branched and transparent. Spore-forming cells were solitary and smooth; conidia were smooth, fusiform or oblong, transparent, blunt-based, mostly erect, and 16.54±2.19 × 3.38±0.77 μm (n=100 in each isolate) in size. Three representative isolates (AB-6, AB-9, AB-16) were selected for further study. For molecular identification, the internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1-α (TEF1), and large subunit ribosomal RNA (LSU) were amplified with primers ITS1/ITS4 (White et al. 1990), EF1-983F/EF1-1567R (Rehner and Buckleyet al. 2005), and LR0R/LR5 (Vilgalys and Hesteret al. 1990), respectively. BLASTn searches showed that the ITS (OM280456, ON026088-89), TEF1 (ON055278-80) and LSU (OM281346, ON026097-98) sequences had the highest similarity to Coniella quercicola strains with: 99% (600/605, 600/605, 600/604) identity for ITS (MH859478.1); 98% (326/333, 327/334, 325/332) identity for TEF1 (KX833698.1); 99% (870/872, 833/834, 830/831) identity for LSU (MH871258.1) of ex-type CBS 904.69. A Neighbor-Joining phylogenetic tree was constructed by combining 3 sequenced loci. Three isolates clustered in the C. quercicola clade with 100% bootstrap support. Thus, based on morphological (Maas et al. 1979; Wang and Lin et al. 2004) and molecular characteristics, the pathogen was identified as C. quercicola. In a pathogenicity test, 20 healthy E. cloeziana seedlings with at least 5 leaves were divided into 4 groups: groups 1-3 were used to inoculate three isolates respectively, and the fourth group acted as control. After surface disinfection with 75% ethanol and wiping with sterile water, tiny wounds were maked made by inoculation needle on each leaf. Fungal culture plugsblocks cut from 3 isolates were placed on wounds in groups 1-3 respectively,. withWarter- agar blockplugs served as control in group 4. The leaves were covered with wet cotton and sealed in airtight bags to retain moisture at room temperature with natural light. After 3 days, light brown lesions were observed in groups 1-3, with no symptoms present in the control group. The pathogenicity test was confirmed by repeating in triplicate and fungi re-isolated from symptomatic leaves were identified as C. quercicola. To our knowledge, this is the first report of leaf blight on E. cloeziana caused by C. quercicola in China. This study increases our understanding of E. cloeziana leaf blight and future research may allow the development of targeted prevention methods for more effective disease controls.

Polygonatum sibiricum Delar. ex Redoute is a plant species used for medicine and food. On one hand, its rhizomes have potential medicinal values such as enhancing immunity, anti-aging, anti-tumor and antibacterial as well as the effects of improving memory and reducing blood lipid and sugar. On the other hand, the rhizomes can also be used as raw materials for drinks, preserves, and health products (Su et al. 2018). The annual demand of P. sibiricum is about 3500-4000 tons in China, and the market demands and the price continue to rise in recent years (Su et al. 2018). In August 2019, there was an outbreak of southern blight in the P. sibiricum planting fields (N30°04'06″, E115°39'47″) of Luotian County in Hubei province of China. Approximately 30% of plants were affected in many fields (333.33 ha). We observed that the surface of the infected rhizome and the surrounding soils were covered with white hyphae and sclerotia. The hyphae gradually extended downward to the rhizomes, causing rhizome rot and leaf yellowing and wilting. Mycelial fragments and sclerotia from ten symptomatic rhizomes were collected in the fields and incubated directly on potato dextrose agar (PDA containing 50 µg/ml kanamycin) at 27℃. The fungal colonies were transferred to PDA after two days of cultivation. The white colonies were formed with fluffy aerial mycelia, which grew radially with an average growth rate of 20.54±0.52 mm/d (n=10). The color of the sclerotia was milky white at first, and then gradually turned to beige and yellow-brown. After two-week-incubation, the sclerotia became dark brown. Most of the sclerotia were spherical or nearly spherical, with round-bulges on the surface. The number of mature sclerotia produced per plate ranged from 8-23 (n=10), and the size ranged from 2.5×3.0 mm to 7.5×13.0 mm (5.95 ± 2.34×7.51 ± 2.88 mm; n=50). In addition, clamp connections were observed under the microscope. For molecular identification, genomic DNA was extracted from isolate HJ-1 using the CTAB method (Mahadevakumar et al. 2018). The internal transcribed spacer (ITS) regions of rDNA were amplified with the primers ITS1/ITS4 (White et al. 1990). The resulting showed ITS sequence (Accession number: MW049362) was 99.66% homology with Sclerotium delphinii according to the GenBank database. In addition, the second largest subunit of RNA polymerase II gene (RBP2) and part of the elongation factor 1-alpha (EF1-α) gene were amplified by using the primers RPB26F/RPB2-7CR (Liu et al. 1999) and EF595F/EF1160R, respectively (Wendland and Kothe 1997). RPB2 gene sequence was deposited in GenBank (Accession number: MW415935), and was 99.53% similarity identity to Athelia rolfsii isolate MSB5-1. TEF-1α sequence was deposited in GenBank (Accession number: MW415934), and was 91.35% similarity to S. delphinii strain Sd_405. Because there are very few reference sequences of RPB2 genes from S. delphinii in GenBank to compare, we choose the ITS and TEF-1α gene sequences to construct the concatenated phylogenetic tree by the neighbor-joining method (Tamura et al. 2013). The results showed that HJ-1 was clustered with S. delphinii isolates selected from NCBI database. Based on morphological and molecular characteristics, the fungus was identified as S. delphinii Welch (teleomorph Athelia rolfsii (Curzi) C.C. Tu & Kimbr). Pathogenicity tests were performed on the healthy leaves, roots, stems and plants (n=3) of P. sibiricum. Each sample was inoculated with one sclerotia produced from a fifteen-day-old colony and there was on wound treatment. These inoculated and control samples (treated with sterile water) were incubated in a moist chamber (25 ± 2 °C, RH 85%) (Mahadevakumar et al. 2018). Typical disease symptoms were apparent on leaves, stems, rhizomes and plants at 4, 6, 5 and 15 days post inoculation, respectively. Fulfilling Koch's postulates, the fungal pathogens were isolated and purified from the inoculated site and were reconfirmed as S. delphinii based on the morphological features. To the best of our knowledge, this is the first report of S. delphinii causing southern blight on P. sibiricum in China. S. delphinii has a wide host range worldwide and often causes crop yield reduction. This study will be helpful for the prevention and control of P. sibiricum southern blight in the future.

Roses are an ornamental crop and grown commercially as cut flowers. They are also used in the perfume, jam, and jelly industry. Dieback of rose is a major disease caused by different types of fungi including Lasiodiplodia pseudotheobromae, Acremonium sclerotigenum, Coniothyrium fuckelii, and Botrytis cinerea (Mirtalebi et al. 2016; Wee at al. 2017). Severe peduncle dieback of rose was observed in a garden Sngju, South Korea, in August 2018. Typical symptoms are characterized by progressive death of peduncle from the flower bud. The presumptive causal agent was isolated from infected tissue. Small sections of peel (2 × 2mm) were cut from the margin of infected tissue and disinfected with 0.5% NaOCl for 1 min, followed by rinsing in sterile water. A disinfected section of infected tissue was blotted dry, placed on potato dextrose agar (PDA), and incubated at 25°C in the dark. Two pure isolates (ICKR1 and ICKR2) were obtained by transferring newly emerging hyphal tips to the new fresh PDA plates. Colonies on PDA were creamy white with small black perithecia produced across the colony after 3 weeks. Asci were eight-spored, clavate to cylindrical, and 57.6 to 83.5 × 9.2 to 14.6 μm (mean ± SD = 70.1 ± 9.2 × 12.1 ± 1.35 μm) (n = 35). Ascospores were hyaline, unicellular, curved or straight with round ends, and 15.2 to 30.8 × 5.2 to 8 μm (mean ± SD = 21.4 ± 3.6 × 6.40 ± 0.8 μm) (n = 30). The morphology of asci and ascospores overlapped with the previous morphological description of Glomerella cingulata (teleomorph of C. gloeosporioides) (Ireland et al. 2008; Weir et al. 2012). Only a few conidia were produced on PDA, which were hyaline, cylindrical, straight with one or both ends rounded, and 18.8 to 24.6 × 5.9 to 6.8 μm (mean ± SD = 21.45 ± 1.9 × 6.3 ± 0.45 μm) (n = 20). Appressoria were brown and unlobed. Molecular analysis was reformed using the internal transcribed spacer (ITS) region of rDNA and partial GAPDH, TUB2, CHS-1, ACT, and CAL genes sequence data. The complete ITS region and partial GAPDH, TUB2, CHS-1, ACT, and CAL genes were amplified using ITS1F/ITS4, BT2a/BT2b, ACT-512F/ACT-783R, CHS-79F/CHS-345R, ACT-512F/ACT-783R, and CL1C/CL2C primer sets, respectively (Weir et al. 2012), and sequenced. The resulting sequences were deposited in GenBank (accession nos. LC469311 to LC469322). Phylogenetic analysis (maximum likelihood and neighbor joining) using MEGA 6 software delineated the present isolates from rose as Colletotrichum gloeosporioides. Pathogenicity tests were conducted on small twigs of rose containing at least one bud. Ten twigs were washed in tap water, disinfected with 90% ethanol, rinsed with distilled water, and prepared for inoculation. Each prepared twig was put in a plastic tube containing distilled water. The conidial suspension (1 × 10⁶ conidia /ml) of ICKR1 isolate was sprayed over six twigs. Another four twigs received distilled water and served as control. Both treated and control twigs containing tubes were place in a tube holder rack and incubated at 25°C and 16 h/6 h light/dark. After 12 days, typical dieback symptoms appeared on inoculated twigs, whereas control twigs remained asymptotic. The causal agent was reisolated and identified as C. gloeosporioides and confirmed Koch’s postulates. C. gloeosporioides is a ubiquitous pathogen on a wide range of crops and is responsible for anthracnose. C. gloeosporioides has been reported as the causal agent of Eucalyptus dieback in South Africa and tip dieback of Lygodium microphyllum and L. japonicum in Australia (Ireland et al. 2008; Smith et al. 1998). To our knowledge, this is the first report of dieback of rose caused by C. gloeosporioides in Korea.

Samples of species close to Tremellafibulifera from China and Brazil are studied, and T.fibulifera is confirmed as a species complex including nine species. Five known species (T.cheejenii, T.fibulifera s.s., T. “neofibulifera”, T.lloydiae-candidae and T.olens) and four new species (T.australe, T.guangxiensis, T.latispora and T.subfibulifera) in the complex are recognized based on morphological characteristics, molecular evidence, and geographic distribution. Sequences of eight species of the complex were included in the phylogenetic analyses because T.olens lacks molecular data. The phylogenetic analyses were performed by a combined sequence dataset of the internal transcribed spacer (ITS) and the partial nuclear large subunit rDNA (nLSU), and a combined sequence dataset of the ITS, partial nLSU, the small subunit mitochondrial rRNA gene (mtSSU), the translation elongation factor 1-α (TEF1), the largest and second largest subunits of RNA polymerase II (RPB1 and RPB2). The eight species formed eight independent lineages with robust support in phylogenies based on both datasets. Illustrated description of the six species including Tremellafibulifera s.s., T. “neofibulifera” and four new species, and discussions with their related species, are provided. A table of the comparison of the important characteristics of nine species in the T.fibulifera complex and a key to the whitish species in Tremella s.s. are provided.

Fungal species with a broad distribution may exhibit considerable genetic variation over their geographic ranges. Variation may develop among populations based on geographic isolation, lack of migration, and genetic drift, though this genetic variation may not always be evident when examining phenotypic characters. Fomitopsis pinicola is an abundant saprotrophic fungus found on decaying logs throughout temperate regions of the Northern Hemisphere. Phylogenetic studies have addressed the relationship of F. pinicola to other wood-rotting fungi, but pan-continental variation within F. pinicola has not been addressed using molecular data. While forms found growing on hardwood and softwood hosts exhibit variation in habit and appearance, it is unknown if these forms are genetically distinct. In this study, we generated DNA sequences of the nuc rDNA internal transcribed spacers (ITS), the TEF1 gene encoding translation elongation factor 1-α, and the RPB2 gene encoding the second largest subunit of RNA polymerase II for collections across all major geographic regions where this fungus occurs, with a primary focus on North America. We used Bayesian and maximum likelihood analyses and evaluated the gene trees within the species tree using coalescent methods to elucidate evolutionarily independent lineages. We find that F. pinicola sensu lato encompasses four well-supported, congruent clades: a European clade, southwestern US clade, and two sympatric northern North American clades. Each clade represents distinct species according to phylogenetic and population-genetic species concepts. Morphological data currently available for F. pinicola do not delimit these species, and three of the species are not specific to either hardwood or softwood trees. Originally described from Europe, F. pinicola appears to be restricted to Eurasia. Based on DNA data obtained from an isotype, one well-defined and widespread clade found only in North America represents the recently described Fomitopsis ochracea The remaining two North American clades represent previously undescribed species.

Mungbean, Vigna radia (L.) R. Wilczek, is ranked 2nd next to chickpea (Cicer arietinum) in total cultivation and production in Pakistan. In August of 2022 and 2023, mungbean plants (cv. PRI Mung-2018) were found wilting in a field at the Ayub Agricultural Research Institute, Faisalabad, Pakistan. Wilted leaves turned yellow, died, but remained attached to the stem. Vascular tissue at the base of the stem showed light to dark brown discoloration. Roots were stunted with purplish brown to black discoloration. Symptomatic mungbean plants were collected from fields at five different locations (20 samples/location). Disease incidence was similar among the five fields, ranging from 5 to 10% at each location depending upon type of germplasm and date of sowing. For fungal isolation and morphological identification, symptomatic stem and root tissues were cut into ~5 mm2 pieces with a sterilized blade. Tissues were surface-sterilized for one min in a 0.5% sodium hypochlorite solution, rinsed twice in sterilized water, air dried on sterilized filter paper, and aseptically placed on potato dextrose agar (PDA) containing 0.5 g/L-1 streptomycin sulphate. Plates were incubated for 3-4 days at 25 ± 2°C with a 12-h photoperiod. Single-spore cultures were used for morphological and molecular analyses. Isolates on PDA grew rapidly and produced abundant white aerial mycelium that turned off-white to beige with age. Macroconidia were hyaline, falcate, typically 3-to-6 septate with a pointed apical cell and a foot-shaped basal cell, measuring 24.5-49.5 x 2.7-4.7 μm (n = 40). Globose to obovate chlamydospores measuring 5.8 ± 0.5 μm (n = 40) were produced singly or in chains and were intercalary or terminal and possessed roughened walls. The morphological data indicated the isolates were members of the genus Fusarium (Leslie and Summerell 2006). To obtain a species-level identification, a portion of translation elongation factor 1-α (TEF1), the largest subunit of RNA polymerase (RPB1), and the second largest subunit of RNA polymerase (RPB2) region were PCR amplified and sequenced using EF1/EF2 (O'Donnell et al. 1998), Fa/G2R (Hofstetter et al. 2007), and 5f2/7cr (Liu et al. 1999) primers, respectively. DNA sequences of these genes were deposited in GenBank under accession numbers MW059021, MW059017 and MW059019, respectively. The partial TEF1, RPB1 and RPB2 sequences were queried against the Fusarium MLST database (https://fusarium.mycobank.org/page/Fusarium_identification), using the polyphasic identification tool. The BLASTn search revealed 99.9% identity of the isolate to F. nanum (Xia et al. 2019), formerly FIESC 25 of the F. incarnatum-equiseti species complex (MRC 2610, NRRL 54143; O'Donnell et al. 2018). To confirm pathogenicity, roots of 3-5 leaf stage mungbean seedlings were soaked in a 106 spores ml-1 conidial suspension of the fungus for 15 min and then planted in 10 cm pots containing sterilized soil. Mock-inoculated plants with sterile water served as a negative control. Twenty pots that were used for each inoculated and control treatment were maintained at 25 ± 2°C, 14:8 h photoperiod, and 80% relative humidity in a growth chamber. After 15 days, leaf yellowing, internal browning from the base of stems and root discoloration was observed in all the inoculated plants. The uninoculated negative control plants remained asymptomatic. Fusarium nanum was re-isolated from artificially inoculated plants and identified by colony growth, conidial characteristics on PDA and molecular analyses (TEF1). To our knowledge, this is the first report of wilt caused by F.nanum on mungbean in Pakistan. In Pakistan, mungbean cultivation in irrigated areas has increased in recent years. It has been introduced frequently in citrus orchards, crop rotation of maize and sesame, intercropping with sugarcane and as green manure. However, citrus, maize, sesame and sugarcane are also hosts of Fusarium spp. Therefore, this information warrants sustainable crop protection and may have an impact on further interaction of F. nanum with other wilt pathogens.

Bothriochloa ischaemum (family Poaceae) is a perennial weed that can be found in borders of agricultural fields, pastures and roadsides in Malaysia. B. ischaemum is an important phytoremediation species in copper tailings dams (Jia et al. 2020). In December 2021, chlorotic spots with brown halos were observed on leaf samples of B. ischaemum with an incidence of approximately 80% in Penampang, Sabah province (5°56'50.4"N, 116°04'32.8"E). On older leaves, the spots coalesced into larger chlorotic spots. Small pieces (5 x 5 mm) of infected leaves collected from three plants were excised, and then surface sterilized according to Khoo et al. (2022). The fungus was isolated (one isolate was obtained) and cultured on potato dextrose agar (PDA) at 25°C. After 3 days, the colony had cottony aerial mycelia with light purple concentric rings appearing on the underside of the colony. Chlamydospores were produced, either unicellular or multicellular. Conidia were unicellular, hyaline, oval, and were 3.7 to 5.1 x 1.8 to 2.6 μm (n=20). Pycnidia were spheroid, and were 66.4 to 115.3 x 43.1 to 87.4 μm (n=20). Genomic DNA was extracted from fresh mycelia of the fungus based on the extraction method described by Khoo et al. (2022). Amplification of the internal transcribed spacer (ITS) region and large subunit (LSU) of rDNA, and actin (ACT), tubulin (TUB) and RNA polymerase II second largest subunit (RPB2) genes was performed using ITS1/ITS4, LR0R/LR7, ACT512F/ACT783R, T10/Bt2b and RPB2-5F2/RPB2-7cR primers, respectively (O'Donnell and Cigelnik, 1997; Liu et al. 1999; Sung et al. 2007; Chen et al. 2021). The PCR products were sequenced at Apical Scientific Sdn. Bhd.. Sequences were deposited in GenBank as OM453926 (ITS), OM453925 (LSU), OM451236 (ACT), OM451237 (TUB) and OM863567 (RPB2). Sequences of our isolate had 100% homology to ITS of isolate UMS (OK626271) (507/507 bp), LSU of isolate UMS (OM238129) (1328/1328 bp), ACT of isolate CZ01 (MN956831) (275/275 bp), TUB of isolate BJ-F1 (MF987525) (556/556 bp) and RPB2 of isolate HYCX2 (MK836295) (596/596 bp) sequences. Phylogenetic analysis was performed using the maximum likelihood method based on the general time reversible model with a gamma distribution and invariant sites (GTR + G + I) generated from the combined ITS, TUB, LSU and RPB2 sequences, indicating that the isolates formed a supported clade to the related Epicoccum sorghinum type sequences. Morphological and molecular characterization matched the description of E. sorghinum (Li et al. 2020). Koch's postulates were performed by spray inoculation (106 spores/ml) on the leaves of three healthy B. ischaemum plants, using isolate BPL01, while sterilized water was sprayed on three additional B. ischaemum which served as the control. Symptoms similar to those occurred after 6 days post inoculation. No symptoms occurred on controls. The experiment was repeated two more times. The reisolated pathogen was morphologically and genetically identical to E. sorghinum. E. sorghinum was reported previously on Brassica parachinensis (Yu et al. 2019), Camellia sinensis (Bao et al. 2019), Myrica rubra (Li et al. 2020), Oryza sativa (Liu et al. 2020) and Zea mays (Chen et al. 2021) in China. To our knowledge, this is the first report of E. sorghinum causing leaf spot on B. ischaemum in Malaysia. Our findings expand the geographic range and host range of E. sorghinum in Malaysia. B. ischaemum which is a weed in agricultural fields is a host of the pathogen and therefore could be a potential threat to Brassica parachinensis, Camellia sinensis, Oryza sativa and Zea mays in Malaysia. Weed management could be an effective way to eliminate inoculum sources of E. sorghinum.

Species of Neocosmospora are commonly found in soil, plant debris, and living woody or herbaceous substrates and occasionally found in water and air. Some species are reported as saprobes, endophytes, opportunistic pathogens of plants and animals, or producers of bioactive natural products, cytotoxic compounds, and industrial enzymes. To reveal the species diversity of Neocosmospora, specimens from different provinces of China were investigated. Five new species, Neocosmospora anhuiensis, N. aurantia, N. dimorpha, N. galbana, and N. maoershanica, were introduced based on morphological characteristics and DNA sequence analyses of combined calmodulin (CAM), the internal transcribed spacer (ITS), the second largest subunit of RNA polymerase II (RPB2), and the translation elongation factor 1-α (TEF1) regions. Differences between these new species and their close relatives are compared in detail.

The areca palm (Areca catechu L.), belonging to the Palmaceae family, is an important economic crop in Hainan. In June 2018, symptoms of leaf spot were observed on nearly 20% of A. catechu in Qionghai, Hainan (19°40′N; 110°33′E). At first, leaves exhibited small yellow spots; as symptoms progressed, the middle of the lesions appeared black with distinct yellow halos. Lesions were long oval, sometimes irregular with black to brown, small black spots and distinct yellow halos. As the lesions continued to expand, necrotic spots enlarged, gradually gray, and then combined to form larger necrotic areas. Ten leaves with typical symptoms were randomly collected. Small tissue sections (5 × 5 mm) were excised from the margins of lesions, disinfected with 75% ethanol for 10 s and 1% sodium hypochlorite for 2 min, followed by a triple wash with sterile water, plated on PDA, and incubated at 28°C. Three days after incubation, fast-growing fungal colonies with white mycelia appeared. Incubation continued for 3 weeks and few pycnidia developed. Alpha conidia were 5.9 to 8.5 × 2.3 to 3.0 µm (avg. 7.3 × 2.7 µm, n = 100), aseptate, hyaline, fusiform, and acute at both ends. Beta conidia were 14.8 to 29.6 × 1.3 to 2.1 µm (avg. 22.5 × 1.7 µm, n = 100), hyaline, filiform, curved, and tapering toward both ends. These morphological characteristics were similar to Diaporthe spp. (Guarnaccia and Crous 2017). To further confirm the identity of the isolate, single-spore isolates were cultured on PDA and selected for DNA extraction. The internal transcribed spacer (ITS) gene was amplified using primers ITS1 and ITS4 (White et al. 1990), a partial sequence of β-tubulin (TUB) gene by T1 (O’Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995), translation elongation factor 1-α (TEF1) gene by EF1-728F/EF1-986R, and calmodulin (CAL) by CAL-228F/CAL-737R (Carbone and Kohn 1999); sequence data were deposited in GenBank (MN424525, MN424539, MN424567, MN424581). BLAST analysis demonstrated that these sequences were 99% similar to the ITS (MF418423), TUB (MF418583), TEF1 (MF418502), and CAL (MF418257) of D. limonicola. Moreover, a phylogenetic tree was constructed using the method of maximum likelihood (MEGA7.0 (Kumar et al. 2016)) with a combined dataset of ITS, TUB, TEF1, and CAL sequences, which clustered the isolate with D. limonicola (CPC 31137) with high bootstrap support (100%). Based on the morphology, sequence data, and phylogenetic analysis, these isolates were determined as D. limonicola. To validate the results, a pathogenicity test was performed. Ten healthy leaves were washed with sterile water, and each leaf was slightly punctured eight times by a sterile pin and divided into two groups. The first group was placed on a mycelial disk taken from the edges of 4-day-old PDA cultures on each wounded leaf, whereas the other group was placed on PDA agar as a control. All plants were incubated at 28 ± 2°C and 100% relative humidity. Seven days after inoculation, leaves placed on the mycelial disk exhibited the typical symptoms (i.e., small black spots and gray to brown lesions surrounded by yellow halos), whereas controls showed no symptoms. Further, D. limonicola was reisolated from the leaf spot. Pathogenicity tests were repeated thrice with the same results. Koch’s postulate was achieved by the reisolation of D. limonicola from the inoculated leaves. D. limonicola has previously been reported to cause trunk canker of Citrus limon and C. sinensis in Italy (Guarnaccia and Crous 2017). To our knowledge, this is the first report of D. limonicola causing leaf spot on A. catechu in China.

From 2018 to 2020, powdery mildew-like signs and symptoms were observed on chayote (Sechium edule var. virens levis) in a commercial field located in Santa María del Río, San Luis Potosí, Mexico. Signs appeared as whitish powdery masses on both sides of leaves and stems. Disease incidence was about 30% and signs covered up to 70% of leaf surface. Ten samples were collected and analyzed. Mycelium was amphigenous, persistent, white, in dense patches. Hyphal appressoria were lobed and solitary. Conidiophores (n = 30) were hyaline, erect, straight, and 62 to 101 μm long. Foot cells were cylindrical and straight, followed by 1-3 shorter cells, and forming conidia in short chains. Conidia (n = 100) were hyaline, surface striate, cylindrical-ellipsoid, doliiform or ovoid, 25.7 to 37.6 × 11.9 to 18.4 μm, without fibrosin bodies, and with germ tubes terminal or subterminal. Conidial appressoria were lobed. Chasmothecia were not observed. The morphological characters were consistent with those of the anamorphic state of Neoerysiphe sechii (Gregorio-Cipriano et al. 2020). A voucher specimen was deposited in the Herbarium of the Department of Agricultural Parasitology at the Chapingo Autonomous University under accession number UACH192. To confirm the identification of the fungus, genomic DNA was extracted from conidia and mycelium, and the internal transcribed spacer (ITS) region and part of the 28S gene were amplified by PCR and sequenced. The ITS region of rDNA was amplified using the primers ITS5/ITS4 (White et al. 1990). For amplification of the 28S rRNA partial gene, a nested PCR was performed using the primer sets PM3 (Takamatsu and Kano 2001)/TW14 (Mori et al. 2000) and NL1/TW14 (Mori et al. 2000) for the first and second reactions, respectively. Phylogenetic analyses using the maximum parsimony and maximum likelihood methods, including ITS and 28S sequences of isolates of Neoerysiphe spp. were performed and confirmed the results obtained in the morphological analysis. The isolate UACH192 grouped in a clade with isolates of N. sechii. The ITS + 28S sequence was deposited in GenBank under accession number MZ468642. Pathogenicity was confirmed by gently dusting conidia from infected leaves onto ten leaves of healthy chayote plants. Five non-inoculated leaves served as controls. The plants were maintained in a greenhouse at 25 to 30 ºC, and relative humidity of 60 to 70%. All inoculated leaves developed similar symptoms to the original observation after 8 days, whereas control leaves remained disease free. Microscopic examination of the fungus on inoculated leaves showed that it was morphologically identical to that originally observed. The pathogenicity test was repeated twice with similar results. Based on morphological data and phylogenetic analysis, as well as pathogenicity test, the fungus was identified as N. sechii. This pathogen has been previously reported causing powdery mildew on S. edule and S. mexicanum in Veracruz, Mexico (Gregorio-Cipriano et al. 2020). However, to our knowledge, this is the first report of N. sechii causing powdery mildew on chayote in San Luis Potosí (Central Mexico). This pathogen represents a serious threat to chayote production and disease management strategies should be developed.

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

Root rot is an important disease of tea plants owing to its unobvious early symptoms and permanent damage (Huu et al. 2016). In 2019, 5% of tea plants displayed symptoms consistent with root rot in a tea plantation (28°09'N, 113°13'E) located in Changsha city, Hunan province of China. The symptoms of the diseased tea plants ranged from wilting leaves to entirely dead. The roots had black lesions and rot typical of this disease. Symptomatic roots were collected, washed with water and disinfected with 75% ethanol, then cut into pieces and sterilized with 0.1% mercuric chloride for 30 s, 75% ethanol for 1 min, and rinsed with sterile water five times. After drying on sterilized filter paper, root tissues were cultured on potato dextrose agar (PDA) medium at 25 oC for 7 days in the dark. Four isolates, CAGF1, CAGF2, CAGF3, and CAGF4 were purified by selecting single spores. All isolates were subjected to a pathogenicity test. A conidial suspension of each strain was collected at a concentration of 2×106 conidia/mL. For the pathogenicity test, two-year-old field grown tea plants were transplanted in plastic pots containing 240 g of the rice grain-bran mixture (inoculated with 4 mL of conidial suspension and cultured for 14 days) and 960 g of sterilized soil (Huu et al. 2016). The pots without inoculated mixture served as control group. All the pots were kept in illumination incubators at 25 oC and a 12L:12D photoperiod. The pathogenicity test for each strain was repeated three times with three repetitions. Only strain CAGF1 exhibited pathogenicity to tea plants. Symptoms appeared on the third day post inoculation (dpi) and gradually worsened by the 7 dpi. On the 14 dpi, most leaves had died and the roots were black and partially rotten, similar to field symptoms. The reisolated fungus from potted roots was identified as CAGF1 based on ITS region and colony morphology, while isolation was attempted, CAGF1 was not isolated from the control plants, which fulfilled Koch's postulates. On PDA, the colony center of CAGF1 was purple with white margin, while on carnation leaf agar (CLA) medium was white. On CLA medium, macroconidia have 0 to 3 septa, measured 19.1 μm to 41.2 μm × 4.2 μm to 5.4 μm (mean= 31.2 μm × 4.8 μm, n=30). The microconidia were measured as 6.7 μm to 12.8 μm × 2.4 μm to 4.9 μm (mean= 10.1 μm × 3.3 μm, n=30), with 0 to 1 septa. And the chlamydospores were measured as 6.0 to 9.7μm (mean= 7.7μm, n=30). Morphologically, strain CAGF1 was identified as Fusarium oxysporum (Leslie and Summerell 2006). Additionally, the genomic DNA of strain CAGF1 was extracted by cetyltrimethylammonium bromide (CTAB) method, the internal transcribed spacer (ITS), elongation factor 1 alpha (EF-1α) and second largest subunit of RNA polymerase II (RPB2) were amplified using the primers ITS1/ITS4 (White et al. 1990), EF-1/EF-2 (Geiser et al. 2004) and fRPB2-5F/fRPB2-7cR (Liu et al. 1999), respectively. Sequences were deposited in GenBank (ITS, OK178562.1; EF-1α, OK598121.1; RPB2, OP381476.1). BLASTn searches revealed that strain CAGF1 was 100% (ON075522.1 for ITS and JX885464.1 for RPB2) and 99.6% (JQ965440.1 for EF-1α) identical to Fusarium oxysporum species complex (FOSC). Based on phylogenetic analysis, the strain CAGF1 was identified as Fusarium cugenangense, belonging to FOSC. To our knowledge, this is the first report of F. cugenangense causing root rot of tea plants in China. The findings are important for the management of this root rot and the improvement of economic benefits of tea cultivation.