Translation Elongation Factor 1-alpha Sequences Research Articles

First Report in China of Leaf Blight on Spatholobus suberectus Dunn Caused by Diaporthe tulliensis.

Spatholobus suberectus Dunn is an important Chinese medicinal plant, which is the only officially recognized authentic source of the Chinese herbal medicine Spatholobi caulis in the China Pharmacopeia (Qin et al. 2024). In November 2023, a serious leaf disease was observed in the S. suberectus planting base in Nanning city (108.22E, 22.51N), Guangxi, China, with an incidence rate of 80%. Initially the symptoms appeared in the diseased leaves tips and was irregular in shape. The necrosis displayed discoloration of the gray color with a deep brown margin and this gradually expanded through into the lamina. To isolate the causal agent, 10 symptomatic leaves were collected and small segments (5×5 mm) of infected tissues were cut, and then they were surface sterilized (30s in 75% EtOH, 2 min in 2.5% NaOCl, and rinsed three times in sterilized distilled water), placed on potato dextrose agar (PDA) and incubated at 28℃ for 5 days. The hyphal tips were transferred to PDA to obtain pure cultures. 36 isolates were obtained, 23 of them had similar morphology, which were isolated from all the symptomatic leaves and were considered pathogens, and named YY-3-S. After 7 d, the colonies were white, fluffy, with white aerial mycelium. Sporulation was induced using pine needle medium (Su et al. 2012). After 30 days, the globose pycnidia developed, which were superficial to semi-immersed on the pine needle medium, and they appeared to be orange to black in color, with pale yellow conidial droplets. The alpha conidia spores were 3.53 to 6.85×1.75 to 3.70 μm (n = 50). They were unicellular, hyaline, aseptate and ellipsoidal to fusiform. These morphological features were consistent with Diaporthe sp (Sun et al. 2021). For further molecular identification, isolate YY-3-S underwent sequencing of the internal transcribed spacer (ITS), beta-tubulin (TUB2), and translation elongation factor 1-alpha (TEF1) sections (Huang et al. 2021). Obtained sequences of ITS, TUB2 and TEF1 (Genebank accessions nos. PP838571, PP865050 and PP865051) displayed a 99% similarity to Diaporthe tulliensis (Genebank accessions nos. OP163562, KR936132, OR662168, respectively). Phylogenetic analysis was based on a combination of ITS, TUB2 and TEF1 sequence data using MEGA 11 software to construct a phylogenetic tree with Neighbor-Joining. In the phylogenetic tree, the combined sequences attributed YY-3-S to D. tulliensis. The pathogenicity test was conducted on 1-year-old S. suberectus seedlings. We sprayed the mixed suspension of spores and mycelia on the unwounded leaves, while control seedlings received sterile water. 3 leaves per plant and 5 plants per treatment were selected for assessment. All seedings were placed in pots in a moist chamber at 28°C and 90% humidity. After 5 d, browning was observed on all the inoculated leaves tips. 14 d after inoculation, necrotic lesions expanded outward and their symptoms like those in the field, while control plants remained healthy. D. tulliensis was reisolated from the inoculated leaves, fulfilling the Koch's postulates. D. tulliensis has been reported to necrotic spots and leaf blight on Rambutan (Serrato-Diaz et al. 2023), leaf spot on bodhi tree (Li et al. 2022), stem canker on Jasmin (Hsu et al. 2023). To our knowledge, this is the first report of D. tulliensis causing leaf blight on S. suberectus in China. It is important to ensure timely management of the newly emerging disease.

Identification and Fungicide Sensitivity of Fusarium spp. Associated with Root Rot of Scutellaria baicalensis in Shanxi Province, China.

Fusarium root rot is usually classified as an extremely destructive soilborne disease. From 2020 to 2021, Fusarium root rot was observed in production areas and seriously affected the yield and quality of Scutellaria baicalensis in Shanxi Province, China. Based on morphological characteristics and combined analysis of the internal transcribed spacer region of ribosomal DNA and translation elongation factor 1-alpha sequences, 68 Fusarium isolates obtained in this work were identified as F. oxysporum (52.94%), F. acuminatum (20.59%), F. solani (16.17%), F. proliferatum (5.88%), F. incarnatum (2.94%), and F. brachygibbosum (1.47%). In the pathogenicity tests, all Fusarium isolates could infect S. baicalensis roots, presenting different pathogenic ability. Among these isolates, F. oxysporum was found to have the highest virulence on S. baicalensis roots, followed by F. acuminatum, F. solani, F.proliferatum, F. brachygibbosum, and F.incarnatum. According to fungicide sensitivity tests, Fusarium isolates were more sensitive to fludioxonil and difenoconazole, followed by carbendazim, thiophanate-methyl, and hymexazol. In brief, this is the first report of Fusarium species (F. oxysporum, F. acuminatum, F. solani, F. proliferatum, F. incarnatum, and F. brachygibbosum) as causal agents of root rot of S.baicalensis in Shanxi Province, China. The fungicide sensitivity results will be helpful for formulating management strategies of S. baicalensis root rot.

First Report of Panax notoginseng Leaf spot Disease Caused by Boeremia exigua in China.

Panax notoginseng a perennial herb native to China, is widely grown in the Yunnan Province. (Yang et al. 2022). From July to August 2022, a new leaf spot disease was observed on fully expanded leaves of P. notoginseng from a planting base in the Xundian, Yunnan Province, China. Approximately 250 ha. of P. notoginseng is the cultivated area, and the incidence of leaf spot disease was around 10-15%. Round spots appeared on the infected leaves and as the disease progressed these leaves fell off the plant. A total 21 symptomatic leaves were randomly collected from the planting base to isolate the pathogens and further study in the laboratory. The surface of infected leaves were sanitized with 0.5% sodium hypochlorite for 2 min. and 75% alcohol for 1 min., and then rinsed thrice with sterile water. Once drying, the samples were placed on potato dextrose agar (PDA), plates and incubated at 25 °C for 5 days. The fungus was isolated from the symptomatic tissue, but only three isolates were preserved for further identification. Pure cultures of the representative strain Zhaochanglin 118 were obtained using the singlespore method, and the colonies obtained were dark-green to dark-black in appearance. The pycnidia were dark brown, solitary, or congregated with an inconspicuous neck. The conidia were colorless, ellipsoidal, and measured between 4.5 to 7 × 2 to 3 μm (n = 30). These morphological characteristics were similar to those described for Boeremia exigua (Valenzuela-Lopezi et al. 2018). The genomic DNA of the isolate was extracted using the DN14 cetyltrimethylammonium bromide rapid plant genome extraction kit. The internal transcribed spacer (ITS), RNA polymerase second largest subunit (RPB2) and translation elongation factor 1-alpha (TEF1) genes were amplified via polymerase chain reaction using the primers ITS1/ITS4 (White et al. 1990), Af/Cf (Matheny et al. 2002), and EF1-983F/EF1-2218R (Chen et al. 2015), respectively. All sequences were deposited in GenBank (OQ996531 for ITS; OR291158 for RPB2 and OR291159 for TEF1). A BLASTN homology search using the ITS nucleotide sequence indicated that this has 99.6% identity with the sequence MH859059, named B. exigua from CBS culture collection (517/519 bp); the RPB2 sequence has 97.5% identity with sequence GU371780, named B. exigua from CBS culture collection (704/722 bp); and the TEF1 sequence has 98.4% identity with sequence GU349080, named B. exigua from CBS culture collection (871/885 bp). To test Koch's postulates, a pathogenicity test was carried out on the leaves of six fully expanded P. notoginseng plants in the Xundian planting base. Conidial suspensions were prepared for one isolates at a concentration of 106 spores per milliliter. Three leaflets on different plants were applied with 20µl spore suspension and the other three leaflets were drop of 20 µl sterile distilled water. The whole experiment was repeated three times. The P. notoginseng plants were incubated under sterile conditions at 25°C for 7 days. Inoculated leaves showed the characteristic brown round spots, while control leaves were asymptomatic so, Koch's postulates were fulfilled by re-isolating the pathogen from symptomatic tissue, which was subsequently confirmed as B. exigua through morphological and molecular analyses. Koch's postulates were fulfilled. To our knowledge, this is the first report of B. exigua causing leaf spot disease in P. notoginseng in China, which lays a foundation for further study and developing disease control methods.

First report of stem gray blight on Hylocereus megalanthus and Hylocereus polyrhizus caused by Diaporthe arecae in Brazil.

In November 2021, stem gray blight symptoms were seen on two dragon fruit (pitaya) species (Hylocereus megalanthus and H. polyrhizus) in an orchard with 100% disease incidence in Fortaleza, Ceará, Brazil (3°44'24.5"S 38°34'30.8"W). The symptoms were initially yellowish to dark brown lesions, and as the symptoms progressed, the lesions turned grayish with small black pycnidia in the center. Isolation was carried out by disinfecting small pieces of the symptomatic stems in 70% ethanol for 1 min, followed by 1% NaOCl for 1 min, and then rinsed three times with sterile distilled water. Excess water was removed using sterile filter paper. Then the stem fragments were placed on PDA media. Colonies produced small black pycnidia with conidia and some were sterile after 68 days of incubation. Two monosporic isolates were obtained from the colonies: UFCM 0708 from H. megalanthus and the UFCM 0710 from H. polyrhizus, which were used for pathogenicity test, morphological and molecular identification. The colony on PDA was smoke gray with aerial mycelium and the reverse was smoke grey to dark grey. The α-conidia from UFCM 0708 and UFCM 0710 were hyaline, aseptate and fusiform and measured 6.4 to 9.7 (8.0) x 1.2 to 2.4 (1.7) µm and 6 to 13.1 (8.2) x 1.7 to 2.4 (2.0) µm, respectively. The β-conidia from UFCM 0708 and UFCM 0710 were hyaline, aseptate and filiform and measured 15 to 22.5 (18.8) x 0.6 to 1.7 (1.0) µm, and 17.2 to 27.5 (22.3) x 0.5 to 1.0 (0.8) µm (n=30), respectively. This morphology placed the isolates as Diaporthe sp. (Udayanga et al. 2012). For further confirmation, genomic DNA was extracted from the isolates (UFCM 0708 and UFCM 0710), and beta-tubulin (TUB2) and translation elongation factor 1-alpha (TEF1) gene fragments were amplified. BLASTn search results with isolates TEF1 and TUB2 sequences varied from 98.58% to 99.52% identity to the ex-type sequence of Diaporthe arecae (CBS 161.64). Phylogenetic analysis of concatenated sequences alignment carried out using the Maxinum-likelihood and Bayesian Inference analysis placed the isolates within D. arecae clade with 86% bootstrap and 0.99 posterior probabilities support. The sequences obtained in this study were deposited in GenBank (TEF1: OP534720 and OP534722; TUB2: OP534717 and OP534719). The isolates were confirmed as D. arecae based on molecular analysis and morphological characteristics (Gomes et al. 2013). Koch's postulates were completed as described by Karim et al. (2019) through the inoculation of six stems of each dragon fruit (pitaya) species. The stems were wounded by removing a 5 mm diameter disc and after that they were inoculated with a 5 mm diameter mycelial plug from 5 days old PDA plates. PDA plugs were used as control. Each stem was covered with a plastic bag and sterilized water was added into the sterilized filter paper to maintain humidity. The bags were kept in a room at day and night temperature of 25 ± 2 °C. The same symptoms seen in the field appeared on the stems 21 days after inoculation. The control stems remained symptomless. Diaporthe arecae have been reported on H. polyrhizus in Malaysia (Huda-Shakirah et al. 2021). To our knowledge, this is the first report of D. arecae on H. megalanthus and H. polyrhizus in Brazil.

First Report of Nigrospora Species Causing Leaf Spot on Olive (Olea europaea L.)

Leaf spot symptoms were spotted in two olive orchards in Istria and in Kvarner Gulf, Croatia. Fungal species from three representative isolates (P13 LECIII, R18 BI, JA20 NP) have been morphologically characterized based on the colony and conidial characteristics. Several techniques were performed for inducing the sporulation of the JA20 NP isolate. Only PDA + banana medium was successful. PCR was conducted for ITS, TUB, and EF1α gene regions. Phylogenetic analyses were performed using internal transcribed spacer, beta-tubulin, and translation elongation factor 1-alpha sequence data. Three types of tests were conducted: a pathogenicity test on detached leaves, on detached and scratched leaves, and on olive seedlings. Ultimately, from the morphological characterizations, DNA sequence analysis of ITS, TUB, and EF1α gene regions, and phylogenetic analysis, these species were identified as Nigrospora gorlenkoana Novobr., Nigrospora osmanthi Mei Wang & L. Cai, and Nigrospora philosophiae-doctoris M. Raza, Qian Chen & L. Cai. This is the first report of Nigrospora species causing leaf spot on olive trees and the first report of Nigrospora philosophiae-doctoris as a plant pathogen. Fungal leaf diseases in conditions that are favorable for infection and disease development can lead to a decrease in the yield and olive oil quality. Therefore, it is necessary to conduct further research and the monitoring of fungal leaf diseases.

Potential role of Fusarium spp. in grapevine decline

Despite studies associating Fusarium spp. with grapevine decline since late 1970s, no consensus has been reached regarding the roles these fungi play in grapevine health. Recent studies in British Columbia, Canada, assessed prevalence of grapevine trunk diseases (GTDs) in young and mature vineyards, and the presence of GTD fungi in ready-to-plant nursery material sold in Canada. This study characterized the Fusarium spp. isolated from grapevines in BC by sequencing part of the translation elongation factor 1-alpha (TEF1) gene, and carried out pathogenicity studies to determine whether Fusarium plays a role in grapevine decline. Fusarium spp. were isolated from 9.8% of samples collected from young vines (≤ 8-year-old), and 7.3% from mature vines (> 8-year-old), showing decline symptoms in commercial vineyards. Fusarium was also isolated from 43.9% of ready-to-plant dormant grapevines analyzed from four nurseries. Fusarium incidence varied between plants within the same nursery and between plants from the different nurseries. DNA sequences of TEF1 allowed identification of Fusarium oxysporum, F. proliferatum, F. ramigenum, and a Fusarium sp. Pathogenicity studies were conducted in 1-year-old dormant rooted ‘Chardonnay’ plants grafted onto ‘3309C’ rootstock, and treatments included: i) whole plant, ii) trimming of roots, iii) cut at rootstock basal ends, and iv) trimming of roots plus cut at rootstock basal ends. Plants were inoculated using standardized methods, and were then planted in a greenhouse. Fusarium was compared with Dactylonectria macrodidyma, D. pauciseptata and Ilyonectria liriodendri used as positive controls. The Fusarium spp. caused necroses in rootstock roots and basal ends that were similar to those caused by black-foot fungi. Fusarium spp. and black-foot fungi reduced root and shoot dry weights when compared with non-inoculated controls, but no statistically significant differences were recorded for most treatments. This study is the first in Canada to identify Fusarium spp. from grapevines. Though Fusarium was common in these grapevines, pathogenicity tests suggest that the identified Fusarium spp. were weakly pathogenic to ‘3309C’ rootstock grapevines.

First report of Didymella macrostoma causing calyx-end rot of pear (Pyrus communis) in The Netherlands.

Pear (Pyrus communis) is an important fruit crop in the Netherlands, with a total production of 400,000 tons in 2020, and 'Conference' is the main pear cultivar that comprises 80% of total pear production area. In the Netherlands, pears are kept in controlled atmosphere cold storage (-0.5°C) up to 11 months after harvest. Calyx-end rot incidences of 1% to 5% were observed on 'Conference' pears from different orchards in surveys from 2019-2021 in packing houses in the Netherlands. Infections showed 1 to 3 cm brown necrosis. Lesions were round, slightly sunken and next to or including part of the calyx. To isolate the causal agent, fruit were rinsed with sterile water, lesions were sprayed with 70% ethanol until droplet runoff, the skin was removed aseptically with a scalpel, and tissue under the lesion was isolated and placed onto Potato Dextrose Agar (PDA) (Oxoid, UK). The PDA plates were incubated at 20°C in the dark, and hyphal tip isolates were transferred to fresh PDA plates. Colonies on PDA were rosy-whitish to peach-colored. Colonies grown on oat meal agar (OA) under UV light were peach to red color, aerial mycelium sparse, and produced a pink to salmon colored conidial matrix. Conidia were irregular-ellipsoidal to allantoid, smooth, hyaline and usually with one or several gutulles. Conidia were sometimes one septate and measured 15.2±2.8 x 4.0±0.7 μm (n =14), but mostly aseptate and measured 7.9±1.7 x 3.2±0.6 μm (n =100). The fungus was morphologically identical to Didymella macrostoma (syn. Phoma macrostoma) (Boerema et al. 2004; Hou et al. 2020). The identity of four representative isolates, WURR-206, WURR-223, WURR-227 and WURR-308, from affected pears from four orchards in the Netherlands, was determined by multilocus gene sequencing. To this end, genomic DNA was extracted using the LGC Mag Plant Kit (Berlin, Germany) in combination with the Kingfisher method (Waltham, MA). Sequences of the internal transcribed spacer (ITS) region of ribosomal DNA, the large-subunit rRNA (LSU) region, partial sequences of beta-tubulin (TUB) and the translation elongation factor 1-alpha (TEF1) gene region were amplified with primers ITS1/ITS4 (White et al. 1990), LROR/LR5 (Vilgalys and Hester 1990), Btub2Fd/Btub4Rd (Woudenberg et al. 2009) and EF1-983F/EF1-1567R (Rehner and Buckley 2005), respectively. Sequences were deposited under GenBank accession numbers ON077588-ON077591 (ITS), ON113487-ON113490 (LSU), ON098515-ON098518 (TUB) and ON098519-ON098522 (TEF1). MegaBLAST analysis revealed that the ITS, LSU, TUB sequences matched with 100% identity to culture collection sequences of Didymella macrostoma in GenBank MH854841 (ITS), MH866341 (LSU), MN983895 (TUB). The TEF1 sequences matched with 99.7% identity to TEF sequence of Didymella macrostoma MT454020. Subsequently, Koch's postulates were performed on 10 'Conference' pears per isolate (WURR-206, WURR-223, WURR-227 and WURR-308). Fruits wiped with 70% ethanol were inoculated in pathogenicity tests with an agar disk (5 mm diameter) of D. macrostoma prepared from the actively growing edge of 14-day-old cultures grown on PDA. Inoculated fruits were sealed in plastic bags and were incubated in darkness at 20°C. Typical symptoms appeared 7-10 days after inoculation on all pears. PDA-only controls remained symptomless. Fungal colonies isolated from the lesions and cultured on PDA morphologically resembled the original isolate from the infected pears. The identity of the re-isolations was confirmed as D. macrostoma by sequencing, thus completing Koch's postulates. To the best of our knowledge, this is the first report of D. macrostoma causing calyx-end rot of pears. The identification of this causal agent is important knowledge necessary for developing control measures for postharvest diseases of pear.

First report of leaf spot on passion fruit in China, caused by Alternaria alternata.

Passion fruit (Passiflora edulis Sims), which is native to South America, is an important fruit crop in tropical and subtropical countries. Passion fruit growing areas have increased rapidly in southern China. In 2018 to 2019, circular spots on passion fruit were observed in Shangsi, Guangxi, China (21°15'N, 107°98'E). The disease occurred from June to April of the following year. The disease incidence was generally between 10% to 30%, but could reach up to 50% in purple passion fruit 'Tainong No.1'. The initial lesions on the fruits were small, with a brown center and a greasy margin, and then became sunken and lighter brown with a diameter of about 1 cm in later stages. The spots on the leaves were often surrounded by a yellow halo and turned into larger lesions after coalescence.. Five typical symptomatic fruit and leaves were collected from Shangsi county for the presumed pathogen isolation. Section of the samples were surface sterilized to isolate the fungus on potato dextrose agar (PDA) at 28°C. Five fungal isolates with similar morphology on PDA were obtained by single spore isolation. Colonies at the age of 7 days accompany with flourishing aerial hyphae, showed surface color varying from white to grey. Conidia were ovate or elliptic, light brown to brown, with 2 to 5 diaphragms, 0 to 4 longitudinal-oblique diaphragms, and mostly 8.2 to 36.7 μm × 5.4 to 15.8 μm. The morphology of the fungus resembled Alternaria alternata (Fr.) Keissl (Simmons, 2007). Each of the five isolates (SF-001, SF-002, SF-003, SF-004 and SF-005) was molecularly identified using genomic regions of 18S nrDNA (SSU), 28S nrDNA (LSU), RNA polymerase second largest subunit (RPB2), internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and translation elongation factor 1-alpha (TEF1) (Jayawardena et al., 2019). Nucleotide sequences of SSU (MZ275254, ON055696, ON055697, ON055698 and ON055699), LSU (MZ275253, ON062947, ON062948, ON062949, ON062950), RPB2 (MZ275251, ON055377, ON055378, ON055379 and ON055380), ITS (MW866522, MW866523, ON053451, ON053452 and ON053453), GAPDH (MZ286628, ON055381, ON055382, ON055383 and ON055384) and TEF1 (MZ275255, ON055373, ON055374, ON055375 and ON055376) were deposited in GenBank database. The LSU, GAPDH and TEF1 sequences showed 100% identity with A. alternata in NCBI (KX609773, MK683852 and MK637432, respectively). The SSU, RPB2 and ITS sequences showed 99% identity to A. alternata (U05194, MK605898 and MN856409, respectively). In pathogenicity test (Zhang et al., 2020), 3-month-old grafted 'Tainong No.1' seedlings and mature fruit were used. Five-mm-diameter mycelial plugs taken from 7-day-old PDA colonies of each of 5 isolates were placed on the leaves and fruit that were wounded with a sterilized needle to form 3 pinpricks. Sterile PDA plugs were used as control. Three plants and three fruits were used in each treatment, and the test was repeated twice. The inoculated plants and fruit were kept in plastic bags and grown in a chamber at 28℃. Typical lesions were observed on inoculated plants and fruit after 3 days, but the controls remained healthy. A. alternata was consistently reisolated from these typical lesions. Previously, leaf spot on passion fruit caused by A. alternata has only been recorded in New Zealand (Rheinländer, 2010). To our knowledge, this is the first report of A. alternata (Fr.) Keissl. causing leaf spot on passion fruit in China. The identification of the pathogen may help to take effective management strategies of controlling this disease.

Fusarium equiseti as an Emerging Foliar Pathogen of Lettuce in Greece: Identification and Development of a Real-Time PCR for Quantification of Inoculum in Soil Samples.

Lettuce is the most commonly cultivated leafy vegetable in Greece, available in the market throughout the year. In this study, an emerging foliar disease observed in commercial farms has been associated to the pathogen Fusarium equiseti, a member of the Fusarium incarnatum-equiseti species complex (FIESC). Thirty F. equiseti isolates obtained from symptomatic lettuce plants were identified on the basis of morphology and evaluated for their pathogenicity. The isolates were further characterized using amplification and sequence analysis of the internal transcribed region (ITS-rDNA), and of the translation elongation factor 1-alpha (TEF1-a), calmodulin (CAM), beta-tubulin (Bt), and small subunit (SSU) genes. Moreover, a novel RT-qPCR assay was developed, designing a primer pair and a probe based on the TEF1-a sequences. This assay showed high specificity, amplifying F. equiseti DNA samples, while no amplification product was observed from samples of other common soilborne fungi. The generated RT-qPCR assay could be a useful tool for the detection and quantification of F. equiseti in soil samples deriving from fields cultivated with lettuce and other leafy vegetables, hosts of this specific pathogen.

Molecular Variation and Phylogeny within Fusarium avenaceum and Related Species

Many recent articles feature research on the Fusarium tricinctum species complex (FTSC), and their authors present different ideas on how the isolates of this species complex can be identified at the species level. In previous studies, our aim was to investigate the phylogeny of FTSC strains, which researchers have morphologically identified as Fusarium avenaceum, Fusarium arthrosporioides, and Fusarium anguioides. In the current study, our phylogenetic maximum parsimony and likelihood analyses of the DNA sequences of the translation elongation factor 1-alpha (TEF1) and combined sequences of TEF1 and beta-tubulin (TUB2) supported the existence of at least four main groups among these strains. Main Group I mainly contains F. avenaceum strains, while Main Group II contains two subgroups, one of which primarily includes F. arthrosporioides strains, and the other mainly includes European F. anguioides strains. Main Group III contains strains from different plants that originated from Asia, including two F. anguioides strains. F. avenaceum strains, which are mostly isolated from different trees, form Main Group IV. A fifth group (Main Group V) was only supported by TEF1 sequences. The main groups previously found by us based on TUB2 sequences could be connected to the new species of the FTSC, which were identified based on TEF1 sequences. In addition, we found strains that significantly differ from Main Groups I-V, and we grouped some of them as single, intermediate, or sister groups. All of the main groups of the present work, and some single and intermediate strains, may represent different species of the FTSC, while the two subgroups of Main Group II constitute intraspecific variation. Regardless of whether they belonged to the main groups, all the analysed strains were able to form different enniatins and 2-amino-14,16-dimethyloctadecan-3-ol, but did not produce beauvericin.

FUSARIUM-ID v.3.0: An Updated, Downloadable Resource for Fusarium Species Identification.

Species within Fusarium are of global agricultural, medical, and food/feed safety concern and have been extensively characterized. However, accurate identification of species is challenging and usually requires DNA sequence data. FUSARIUM-ID (http://isolate.fusariumdb.org/blast.php) is a publicly available database designed to support the identification of Fusarium species using sequences of multiple phylogenetically informative loci, especially the highly informative ∼680-bp 5' portion of the translation elongation factor 1-alpha (TEF1) gene that has been adopted as the primary barcoding locus in the genus. However, FUSARIUM-ID v.1.0 and 2.0 had several limitations, including inconsistent metadata annotation for the archived sequences and poor representation of some species complexes and marker loci. Here, we present FUSARIUM-ID v.3.0, which provides the following improvements: (i) additional and updated annotation of metadata for isolates associated with each sequence, (ii) expanded taxon representation in the TEF1 sequence database, (iii) availability of the sequence database as a downloadable file to enable local BLAST queries, and (iv) a tutorial file for users to perform local BLAST searches using either freely available software, such as SequenceServer, BLAST+ executable in the command line, and Galaxy, or the proprietary Geneious software. FUSARIUM-ID will be updated on a regular basis by archiving sequences of TEF1 and other loci from newly identified species and greater in-depth sampling of currently recognized species.

First Report of Leaf Spot on Kidney Bean Caused by Nigrospora oryzae in China

Kidney bean (Phaseolus vulgaris L.) is a legume with high nutritional and economic value. This vegetable crop is widely cultivated in China, providing a year-round supply of young edible pods. In July 2020, a leaf spot disease on kidney bean cultivar 'Dabailong' was observed on a two-hectare field in Longli County (26°16'15.66″ N, 106°48'12″ E), Guizhou Province, China. Disease incidence was estimated to be nearly 50%. Foliar symptoms manifested as black circular spots, surrounded by a yellow halo and accompanied by white mycelium. To identify the pathogen, small portions of tissue (5×5 mm) from margins of leaf spots were cut from 20 symptomatic leaves, surface disinfected with 75% ethanol for 30 s, rinsed two times with sterile distilled water, dried on a sterile filter paper, and incubated on potato dextrose agar (PDA) at 28°C for 3 days. A total of 39 single-spore isolates were obtained. The colonies on PDA were fluffy, changing from white to gray or black with age, and reaching 7-cm diameter in 5 days at 28°C. Conidia were black, globose to subglobose, smooth, solitary, measuring 13.0 to 16.0 × 10.5 to 16.0 µm (n=30). Morphological characteristics were consistent with Nigrospora oryzae. In addition, the rDNA internal transcribed spacer (ITS), large subunit (LSU), β-tubulin (TUB) and translation elongation factor 1-alpha (TEF1) loci were amplified by PCR and sequenced (White et al. 1990, Glass and Donaldson 1995, O'Donnell et al. 1998; Carbone and Kohn 1999). The ITS, LSU, TUB and TEF1 sequences of two isolates, GUCC19-5105 and GUCC19-5192, were submitted to GenBank. BLASTn analysis of these sequences showed >98% homology with those of N. oryzae strain LC 7293 in GenBank (ITS, 99.80% (MZ145361 vs KX985931 - 498/499 bp) and 99.62% (MZ148445 vs KX985931 - 525/527 bp); LSU, 100% (MZ146317 vs KY806236 - 837/837 bp) and 99.76% (MZ148446 vs KY806236 - 847/849 bp); TUB, 98.72% (MZ329335 vs KY019601 - 386/391 bp) and 98.67% (MZ329337 vs KY019601 - 373/378 bp) and TEF1, 98.91% (MZ329336 vs KY019396 - 452/457 bp) and 98.89% (MZ329334 vs KY019396 - 444/449 bp) respectively). The phylogenetic tree of the combined 4 sequences showed that both isolates clustered with N. oryzae. Based on morphological characteristics and the multigene phylogenetic analysis, GUCC19-5105 and GUCC19-5192 isolates were identified as N. oryzae. Pathogenicity tests were performed twice by spraying conidial suspension (1×105 conidia/mL) of the two isolates (GUCC19-5105 and GUCC19-5192) on leaves of ten (five per isolate) healthy 5-week-old kidney bean cultivar 'Dabailong' plants. Two plants sprayed with sterile water served as controls. After inoculation, all the plants were kept moist in plastic bags for 24 hours and incubated in a greenhouse at 25°C for 20 days. Leaf spots similar to those observed in the field were observed 20 days post inoculation, but no lesions were observed on control plants. N. oryzae was reisolated from the infected tissues of inoculated kidney bean plants and the identity of the reisolated pathogen was confirmed as N. oryzae through morphology and sequencing ITS, LSU, TUB and TEF1 loci. In recent years, N. oryzae has been reported to infect a variety of plants such as Aloe vera, Citrullus lanatus and Costus speciosus (Begum et al. 2018; Chen et al. 2019; Sun et al. 2020). To our knowledge, this is the first report of leaf spot disease on kidney bean caused by N. oryzae in the world and provides a basis for diagnosticians and researchers to identify the disease and develop disease management strategies.

First Report of Nigrospora osmanthi Causing Leaf Blight on Orthosiphon stamineus in Malaysia.

Orthosiphon stamineus (Java tea) is a perennial herbaceous plant in the family Lamiaceae and is cultivated extensively in Southeast Asia for its medicinal value (Arifullah et al. 2014). During October 2018, leaf blight symptoms were observed on leaves of ~210 plants O. stamineus grown in experimental plots of a research farm at Faculty of Engineering, Serdang, Selangor, Malaysia (3°00'30.4"N 101°43'19.9"E) with 80% disease incidence. Initial symptoms were brown-to-black lesions on the leaves that enlarged and coalesced until the leaf withered and abscissed. Diseased tissues (4 × 4 mm) of six infected leaves were excised, surface disinfected with 0.5% NaOCl for 1 min, rinsed twice with sterile distilled water, placed onto potato dextrose agar (PDA) plates, and incubated at 25°C with a 12-h photoperiod under fluorescent light for 7 days. A total of six single-spore isolates were obtained from sampled leaves. All isolates exhibited similar morphology and two representative isolates, MK and MK1, were characterized further. Colonies on PDA were initially white then turned dark gray with age and had a pale green underside. Hyphae were branched, septate and hyaline to pale brown. The conidia were one-celled, black, smooth-walled, spherical to subspherical in shape measuring 11.0 μm × 16.5 μm in diameter (n=30) and which are borne on hyaline vesicles at the tip of each conidiophore or formed directly from the mycelia. Based on morphological characteristics, the fungal isolates were identified as Nigrospora osmanthi (Wang et al. 2017). Total genomic DNA of the isolates was extracted from fresh mycelium using DNeasy Plant Mini kit (Qiagen, USA) and the internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF1) and Beta-tubulin (TUB2) gene regions were amplified using ITS5/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999) and Bt-2a/Bt-2b primer set (Glass and Donaldson 1995), respectively. BLASTn analysis showed that the ITS, TEF1 and TUB sequences of the isolates shared 99%-100% identity with Nigrospora osmanthi ex-type strain CGMCC 3.18126 (GenBank accession nos. KX986010, KY019421, KY019461). The sequences of two representative isolates (MK and MK1) were deposited in GenBank (ITS: accession nos. MT645782, MW363019; TEF1: MW366861, MW366862; TUB: MW366863, MW366864). Phylogenetic analysis by the maximum likelihood method based on the concatenated ITS-TEF1-TUB sequences showed that the isolates in this study were clustered in a strongly supported group 98% maximum likelihood with type strain N. osmanthi (Kumar et al. 2016). The pathogenicity of all isolates was confirmed by inoculation on ten healthy leaves of five potted 4-week-old O. stamineus plants using a conidial suspension (1 x 106 spores/ml) produced on 7-days-old PDA cultures. An equal number of plants were sprayed with sterile distilled water only to serve as a control and the treated plants were kept in a growth chamber for 2 weeks at 28 ± 1°C and 95% relative humidity. The experiment was repeated twice. The inoculated leaves developed brown lesions which enlarged into blight symptoms similar to those observed on naturally infected leaves after 5 days of inoculation, while control plants remained healthy. Nigrospora osmanthi was successfully re-isolated from the infected leaves, but not from leaves of non-inoculated control plants, thus satisfying Koch's postulates. . N. osmanthi has been recently reported to cause leaf blight on Ficus pandurata (Liu et al. 2019) and Stenotaphrum secundatum in China (Mei et al. 2019). This disease can cause a significant threat to the cultivation of O. stamineus which has been extensively grown for the production of herbal Java tea. Accurate identification of this pathogen could assist in developing an effective disease management strategy to control this disease.

Nigrospora oryzae Causing Black Leaf Spot Disease of Hibiscus mutabilis in China.

Cotton rose (Hibiscus mutabilis Linn.) is a deciduous shrub native to China. It has been widely cultivated in many provinces in China for its ornamental and ecological value (Shang et al., 2020). In May 2017, an unknown leaf spot symptom was first observed on H. mutabilis at the Chengdu Campus of Sichuan Agricultural University (30°42'31″ N, 103°51'28″ E). The disease occurred from May to September with approximately 81% incidence by field sample survey of 300 plants in Chengdu Greenway. The symptoms at first appeared as irregular black spots on the leaves. Then the lesions grew and coalesced into large, black necrotic areas, which later produced leaf chlorosis and abscission (Fig. 1-A). This disease seriously reduced the ornamental value of H. mutabilis. Forty diseased lesions (4 × 5 mm) were surface sterilized with 75% alcohol for 60 s and 3% NaClO for 45 s, rinsed three times in sterile water, placed onto potato dextrose agar (PDA), and then incubated in a dark at 25°C. From the 7 obtained isolates, 4 isolates exhibited the morphology described as Nigrospora oryzae (Hao et al., 2020). The fungus produced initially circular white colonies, and then the centers turned dark gray or black with age on the PDA. Hyphae were smooth, branched, septate, hyaline, or pale brown. Conidia (N = 100 spores) were abundant, and were solitary, dark-brown to black, smooth, aseptate, and measured 11 to 15 μm in diameter (Fig. 1). DNA was extracted from the fungal colonies using a DNeasyTM Plant Mini Kit (Qiagen). The internally transcribed spacer (ITS), β-tubulin gene (TUB), and translation elongation factor 1-alpha (TEF1) were amplified with primers ITS1/ITS4 (White et al., 1990), BT2A/BT2B (Glass and Donaldson 1995), and EF1-728F/EF1-986R (O'Donnell et al., 1998; Carbone and Kohn 1999), respectively. BLAST results indicated that the ITS, TUB, and TEF1 sequences (GenBank accession Nos. MN515070, MN733956, and MN635723, respectively) had 99% identity with N. oryzae sequences (GenBank accession Nos. KX986031, KY019553, and KY019358). The result was confirmed by multilocus phylogenetic analysis (Fig. 2). The morphological characteristics and molecular analyses of the isolate matched the description of N. oryzae. To confirm pathogenicity, Koch's postulates were fulfilled under controlle conditions. The seedlings of 20 two-year-old potted H. mutabilis plants were inoculated by spraying conidial suspension at the concentration of 1 × 106 conidia/ml on both sides of leaves. Sterilized distilled water (20 seedlings) were used as negative controls. The experiment was performed three times. All plants were incubated at 25°C ± 2°C under a 16 h/8 h photoperiod and 70%-75% relative humidity (RH) after inoculation, and observed daily for disease development. Two weeks later, the inoculated plants showed the same symptoms as the original diseased plants and the controls remained asymptomatic. The pathogen N. oryzae was re-isolated from all ioculated plants, and the culture and fungus characteristics were the same as those of the original isolate. But N. oryzae was not isolated from the control plants. The results indicated that N. oryzae is a causal agent of the disease. N. oryzae was reported as a leaf pathogen on cotton (Zhang et al., 2012), but this is the first report of N. oryzae causing leaf black spot on H. mutabilis in the world. The identification could provide relevant information for adopting appropriate management strategies to control the disease.

Phylogenetic assessment and taxonomic revision of Halobyssothecium and Lentithecium (Lentitheciaceae, Pleosporales)

Our studies on lignicolous aquatic fungi in Thailand, Sweden, and the UK resulted in the collection of three new Halobyssothecium species (H. bambusicola, H. phragmitis, H. versicolor) assigned to Lentitheciaceae (Pleosporales, Dothideomycetes). Multi-loci phylogenetic analyses of the combined large subunit, small subunit, internal transcribed spacers of ribosomal DNA, and the translation elongation factor 1-alpha sequence data enabled a revision of the taxa assigned to Lentithecium and the transfer of L. cangshanense, L. carbonneanum, L. kunmingense, L. unicellulare, and L. voraginesporum to Halobyssothecium. Collection of an asexual morph of L. lineare and phylogenetic analysis confirmed its taxonomic placement in Keissleriella. Detailed descriptions and illustrations of H. bambusicola, H. phragmitis, and H. versicolor are provided.

First Report of Leaf Spot on Lasia spinosa Caused by Fusarium fujikuroi in China

Lasia spinosa (L.) Thwaites (Araceae) is an ornamental medicinal plant grown in tropical and subtropical countries. The leaf extract of L. spinosa has significant anthelmintic efficacy (Yadav and Temjenmongla 2012). During July 2018 and 2019, L. spinosa plants at the Guangxi Botanical Garden of Medicinal Plants of Guangxi province, China, showed leaves with brown spots or irregular white necrotic lesions that were scattered with some shot holes. Sixty-eight plants from a total of 200 plants showed such symptoms. The overall incidence was about 34%. To isolate the pathogen, 15 infected leaves were excised near the margin of necrotic lesions, surface sterilized with 75% ethanol for 1 min, and treated with 0.5% sodium hypochlorite for 10 min followed with three rinses with sterile water. The leaf sections were then plated on potato dextrose agar (PDA) medium and incubated at 28°C. Further, to obtain pure cultures, hyphal tips from recently germinated spores were excised and transferred onto fresh PDA and incubated at 28°C. Ten fungal isolates with similar morphological characteristics were obtained. Colonies on PDA developed white fluffy aerial mycelia and produced abundant conidia 2 weeks later. Microconidia were abundant, oval, clavate, solitary, and hyaline, with an average size of 7.7 × 3.1 μm (n = 46). Macroconidia were mostly three to five septate, slender, almost straight, and sickle shaped, with an average size of 26.7 × 3.6 μm (n = 30). No chlamydospores were observed. These morphological characteristics were consistent with those of Fusarium fujikuroi (Leslie and Summerell 2006). For further identification, five DNA regions of isolate CYA, corresponding to the internal transcribed spacer (ITS), β-tubulin (TUB2), calmodulin (CL), translation elongation factor 1-alpha (TEF1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were PCR amplified and sequenced (Jiang et al. 2018; Weir et al. 2012). BLASTN analyses of ITS and CL sequences showed 100% similarity with corresponding sequences of F. fujikuroi accession numbers MK630103.1 and MF984412.1; the TUB2 and TEF1 sequences shared 99.71% identity (MH398245.1 and MH341224.1), and the GAPDH sequence shared 98.65% identity (CP023092.1). Sequences were submitted to GenBank under the accession numbers MN704565 (ITS), MN490089 (TUB2), MN490091 (CL), MN490092 (TEF1), and MN490090 (GAPDH). According to those morphological and molecular characteristics, the pathogen was identified as F. fujikuroi. Further, to verify the pathogenicity, spore suspensions (10⁵ conidia/ml) were prepared from 10-day-old PDA cultures and inoculated onto leaves of nine 2-month-old L. spinosa plants using the wound/drop inoculation method (10 μl per wound). Three more plants were inoculated with sterile water as controls. All plants were grown in a growth chamber with a temperature of 28°C and 90% humidity. After incubating for 7 days, leaf spot symptoms were observed on the inoculated leaves, and no symptoms were observed on the control plants. F. fujikuroi was reisolated from leaf lesions on the inoculated leaves, thus confirming Koch’s postulates. The pathogenic experiment was subsequently repeated under the same conditions. To our knowledge, this is the first report of F. fujikuroi causing leaf spot of L. spinosa. Severe leaf spot disease on L. spinosa has affected its medicinal and ornamental values, which suggests breeders should take this disease into consideration in L. spinosa breeding programs.

New Endophytic Fusarium spp. from Fraxinus excelsior Leaves in Slovakia.

Fraxinus excelsior is currently suffering from ash dieback disease caused by the fungus Hymenoscyphus fraxineus. Co-occurrence of large numbers of other fungi with endophytic as well as pathogenic lifestyles has been observed on F. excelsior. In this study, new endophytic Fusarium spp. isolate Fraxinus excelsior Fusarium from Slovak territory, associated with F.excelsior, was characterised. Using morphology-based approaches, the fungus was assigned to the genus Fusarium. The isolate formed thick-walled hyaline, fusiform, slightly curved macroconidia, produced from monophialides in sporodochia, pointed at the tip, mostly 3 - 5 septate, occasionally 6 septate, and 40 - 68 × 3.8 - 5.0 µm in size with basal pedicellate cells. For more accurate taxonomic affiliation, molecular sequence analyses of the internal transcribed spacer, translation elongation factor 1-alpha sequences, and partial β-tubulin gene were performed. While the internal transcribed spacer sequence of the isolate showed the highest similarity to the Fraxinus oxysporum species, translation elongation factor 1-alpha and partial β-tubulin sequences were distantly related to the Fraxinus avenaceum species. Based on these data, the Fraxinus excelsior Fusarium isolate could probably be considered a new species of the Fusarium genus.

Histone H3 gene is not a suitable marker to distinguish Alternaria tenuissima from A. alternata affecting potato.

Potato Alternaria leaf blight is one of the economically most important disease in potato production worldwide. A recent study reported a quick method to distinguish main Alternaria pathogens A. tenuissima, A. alternata, and A. solani using partial histone H3 gene sequences. Using this method, our collection of 79 isolates from 8 provinces in China were presumably separated into A. tenussima and A. alternata. But in depth morphological and genetic analysis casted doubt on this identification. Culture morphologies of six presumed A. alternata isolates (PresA_alt) and six presumed A. tenuissima isolates (PresA_ten) were not significantly different. PresA_ten isolates also produced conidia in branched chains which supposed to be A. aternata. Phylogenetic analyses were conducted using internal transcribed spacer region (ITS) and five genes commonly used for species identification including glyceraldehyde-3-phosphate dehydrogenase (GPDH), translation elongation factor 1-alpha (TEF1), β-tubulin, plasma membrane ATPase (ATPase), and calmodulin genes. The results showed that GPDH and TEF1 sequences of PresA_alt and PresA_ten isolates were identical. The 12 isolates did not cluster by presumed species neither by individual or concatenated sequence comparisons. The phylogeny–trait association analysis confirmed that the two group isolates were undistinguishable by those molecular markers. Analysis of histone H3 gene sequences revealed variable intron sequences between PresA_ten and PresA_alt isolates, but the amino acid sequences were identical. Our results indicate that the previously published method to distinguish Alternaria species based on histone H3 gene sequence variation is inaccurate and that the prevalence of A. tenuissima isolates in China was likely overestimated.

Identification of the pathogens causing konjac dry rot in China

Dry rot causes significant losses of konjac during storage in Hubei Province, China. In this study, the pathogens were obtained from konjac and then tested its pathogenicity through tissue isolation. The pathogens were identified using morphological characteristics and translation elongation factor-1 alpha sequences analysis. Four Fusarium species, namely F. meridionale, F. incarnatum, F. sambucinum, and F. solani, were obtained.

Real-time PCR quantification of Fusarium avenaceum in soil and seeds

The pathogenic fungus Fusarium avenaceum infects a broad range of plant hosts across the globe. While primarily soilborne, F. avenaceum can colonize all plant tissues, including buds, seeds, fruits, stems, crowns, and roots, resulting in significant crop yield reductions and economic losses for growers. In addition to its impact on crop productivity, F. avenaceum produces toxic metabolites that can be transferred to humans and livestock through consumption of infected foods. The ability of F. avenaceum to cause seed decay may be utilized to deplete the weed seedbank in soil, an important integrated weed management strategy. We developed a SYBR Green I-based real-time polymerase chain reaction (qPCR) assay to efficiently detect and quantify F. avenaceum in soil, wild oat (Avena fatua L.) seed caryopses, and wild oat seed hulls. The primer pair was designed from the translation elongation factor 1-alpha (TEF1) gene. In silico and wet lab testing were done to assess the ability of the primers to bind TEF1 sequences from Fusarium spp. and common soil fungi. The findings indicated that the primers were specific to F. avenaceum, and also recognized GenBank TEF1 accessions annotated as F. arthrosporioides, which has been listed as a foliar pathogen of wheat in Oregon, and conspecific with F. avenaceum. Standard curves of F. avenaceum DNA diluted with soil, caryopsis, or hull extracts indicated primer amplification efficiency was not significantly affected by PCR inhibitors. This real-time PCR assay effectively assesses the presence and abundance of F. avenaceum and its close relative F. arthrosporioides, if present, in soil and seed tissues. The assay can be used for endpoint PCR as well.