Translation Elongation Factor 1α Research Articles

Diversity and pathogenicity of the Fusarium fungi occurred in soybean mycobiota

The aim of present study was to reveal the species composition of Fusarium fungi found in the soybean mycobiota from the various origins of Russia, as well as to characterize physiological and biochemical properties of isolates. Identi cation of the taxonomic status of 21 Fusarium strains isolated from seeds, stems and roots of soybeans was clari ed according to data using phylogenetic analysis of the nucleotide sequences of the gene of translation elongation factor 1α. Five fungal species have been identi ed: F. oxysporum (11 strains), F. equiseti (4), F. proliferatum (3), F. solani (2), as well one strain of F. commune, which was identi ed for the rst time in soybean mycobiota in Russia. The morphological and cultural characteristics of the isolates and their pathogenicity to leaves of two soybean varieties in the laboratory conditions were also analyzed. At the cultivation of Fusarium fungi on potato- sucrose agar medium, the optimal temperature range for the growth of all isolates has been established as 25-30 °C, at which the largest colony diameter 68,5-74,0 mm was found in F. equiseti and F. commune strains, and the smallest colonies (49,5-55,8 mm) was detected in F. solani strains. Three F. oxysporum strains and one F. commune strain, isolated from the stems, were characterized by high pathogenicity to the leaves of two soybean varieties. These strains caused the necrosis of leaves of the Isidor variety in the range of average length 16,7-21,7 mm, and the necrosis of leaves of the Selecta 201 variety in the range of 10,7-23,3 mm. At the same time, the most of the analyzed Fusarium strains (52-67 % depending on the soybean variety) were non-pathogenic. A high intraspeci c variability of pathogenicity to soybean leaves was noted among F. oxysporum and F. proliferatum strains

Diaporthe actinidiicola: A novel species causing branch canker or dieback of fruit trees in Henan Province, China

AbstractPear (Pyrus), walnut (Juglans) and kiwifruit (Actinidia) are important cash crops in China. Recently, symptoms of branch canker or dieback have been frequently observed in many orchards in Henan Province and have resulted in high losses of fruit production. In this study, symptomatic branches were collected for aetiological isolation from three hosts during 2020 and 2021. Seven isolates from these hosts were identified as one novel species, based on morphological characteristics and phylogenetic analyses of the concatenated sequences of the internal transcribed spacer of the ribosomal DNA (ITS), translation elongation factor 1α (TEF), β‐tubulin (TUB), histone (HIS) and calmodulin (CAL). We proposed the name Diaporthe actinidiicola and completed Koch's postulates to confirm pathogenicity. Host range evaluation indicated that D. actinidiicola could infect all inoculated fruit trees (pear, kiwifruit, walnut, apple [Malus] and peach [Prunus]) and the isolates from different hosts were most virulent on their original host. This study characterizes a novel Diaporthe species from three different hosts in China and contributes useful data for practicable disease management.

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

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

Systemic infection of Bryoria (Lecanoromycetes, Ascomycota) by Athelia (Agaricomycetes, Basidiomycota) in western North America

ABSTRACT Bryoria (Parmeliaceae, Ascomycota) is one of the dominant genera of hair lichens in western North America and is characteristic of high-elevation conifer forest ecosystems. In areas where Bryoria is abundant, it is common to find thalli in which the thalline filaments become conglutinated, forming brittle dead zones. After sampling Bryoria thalli across western Canada and the northwestern United States at different times of the year, we found that this dieback phenomenon is associated with the winter growth of a mold-forming basidiomycete. We report that this fungus belongs to Athelia (Atheliaceae, Basidiomycota), a genus known to contain lichen pathogens, most notably A. arachnoidea. By sequencing a combination of genetic markers—nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS), partial nuc 28S rDNA (28S), and partial translation elongation factor 1-α (TEF1)—paired with morphometric analyses, we reveal the involvement of at least three additional lineages of lichen-associated Athelia and describe one as a new species, A. abscondita. Athelia abscondita is morphologically distinguished from other Athelia species by its basidia and basidiospores, was found to frequently infect members of Bryoria sect. Implexae, and was occasionally on other foliose and fruticose species within Parmeliaceae.

Fusarium diversity from the Golden Gate Highlands National Park.

Members from the genus Fusarium can infect a broad range of plants and threaten agricultural and horticultural production. Studies on the diversity of Fusarium occurring in natural ecosystems have received less attention than the better known phytopathogenic members of the genus. This study identified Fusarium species from soils with low anthropogenic disturbance found in the Golden Gate Highlands National Park (GGHNP), a part of the Drakensberg system in South Africa. Selective techniques were implemented to obtain 257 individual isolates from the selected soil samples for which the translation elongation factor 1α (tef-1α) gene region was sequenced and compared against the Fusarium MLST and FUSARIUM-ID databases. Phylogenetic analyses, based on maximum likelihood and Bayesian inference, were used to determine species diversity in relation to reference isolates. Species level identifications were made within three of the seven species complexes and identified F. brachygibbosum, F. sporotrichioides, F. andiyazi, and F. gaditjirri based on the FUSARIUM-ID database, with F. transvaalense and F. lyarnte identified against the Fusarium MLST database. This indicated highly diverse populations of Fusarium from soils with low anthropogenic disturbance from the Afromontane grassland region found in mountain ranges.

Five new Graphium species from hardwood trees in Poland

ABSTRACT Graphium species form a well-supported monophyletic lineage within the Microascales (Ascomycota). Members of this genus can be found in association with bark beetles, as well as on tree wounds and in soils. During surveys of bark and ambrosia beetle–associated fungi and cavities made by woodpeckers on hardwood trees in Poland, many isolates with an affinity to Graphium were recovered. They were identified based on their morphological characters and sequence data for the internal transcribed spacer (ITS), 28S rDNA, β-tubulin (TUB2), and translation elongation factor 1-α (TEF1) gene regions. The results revealed five new species, described here as G. brachiatum, G. longistipitatum, G. polonicum, G. radicatum, and G. trypophloei.

First Report of Phytophthora occultans Causing Dieback of Buxus sempervirens in the Czech Republic.

In Nov 2011, and then recurrently since Sep 2020, an extensive decline has been recorded in boxwood (Buxus sempervirens), sometimes with several dozens of damaged individuals planted in private gardens and public areas and purchased in amateur markets in the Czech Republic. The leaves of the plants first showed orange-bronze discoloration, then dried and fell off, and the affected plants died. The roots, collars and stems of these plants had dark brown to black necrotic lesions. Phytophthora occultans Man in 't Veld & K. Rosend. was consistently isolated on selective medium PARPNH (Jung et al. 1996) directly from segments of symptomatic collar tissues and from rhododendron leaf pieces used to bait excised roots. On 20% V8 agar (V8A) and on carrot agar (CA), colonies had a stellate pattern. Radial growth at 25°C was 9.4 mm/day on V8A and 5.3 mm/day on CA. The cardinal growth temperatures were min. 7°C, optimum 25 to 27°C, and max. 32°C. The isolates were homothallic and produced on CA colorless globose oogonia ranging from 25.4 to 36.4 µm (n = 40) in diam. Oospores were slightly aplerotic and measured (n = 40) 22.5 to 31.9 µm in diam., with a 0.9 to 1.5 µm thick wall. The antheridia were predominantly paragynous and averaged 11.5 × 9.9 µm (height × width, n = 40). Noncaducous sporangia were obpyriform, ovoid, elongated to irregular and semipapillate, sometimes bipapillate and measured (n = 40) 31.4 to 73.4 × 17.8 to 32.1 µm, and the L:B ratio was 1.9 to 2.0. Chlamydospores and hyphal swellings were not observed. The morphological characteristics resembled those described for P. occultans (Man in't Veld et al. 2015). The isolates were deposited in the Czech Collection of Phytopathogenic Oomycetes (CCPO) under accession nos. 551.11, 1158.20, 1176.21, 1201.21, 1218.21, 1236.21 and 1261.22. For molecular identification, the internal transcribed spacer (ITS) region, cytochrome oxidase subunit 1 gene (COX1), and translation elongation factor-1α (EF) gene from all isolates were amplified and sequenced using the primer pairs ITS4/ITS5 (White et al. 1990), COXF-CIT/COXR-CIT (Man in't Veld et al. 2015), and ELONGF1/ELONGR1 (Kroon et al. 2004), respectively. The resulting sequences of representative isolates P1158.20 and P1176.21 were deposited in GenBank (accession nos. MW750576 and OP326036 for ITS, ON862131 and OP313505 for COX1 and MW762616 and ON862132 for EF). BLASTn searches of GenBank, using the partial ITS, COX1, and EF sequences, revealed 100, 100, and 99% sequence identity, respectively, to P. occultans ex-holotype culture CBS101557 accessions JX978155, JX978156 and KF650770 (Man in't Veld et al. 2015). Concatenated sequences of the three genes were used to conduct a phylogenetic analysis using the maximum likelihood method in MEGA 11 (Tamura et al. 2021). The isolates were identified as P. occultans based on morphology and a multigene phylogenetic analysis. Koch´s postulates were confirmed by a soil infestation test. Healthy 2-year-old B. sempervirens plants were inoculated (9 plants per isolate and control, isolates no. 1158.20, 1176.21, 1261.22) with three 5-mm-diam. V8A mycelial plugs by inserting into the substrate near the collar. Control plants were treated with sterile agar plugs. All plants were kept in a greenhouse at 25°C and exposed to 24 h of flooding up to collar once a week. All inoculated plants showed wilting, collar lesions and root rot occurred after 21 days, while control plants remained healthy. The pathogen was reisolated from infected plants and confirmed by molecular identification. P. occultans was found for the first time in 1998 on Buxus sempervirens in the Netherlands and later in Belgium, the United Kingdom, Germany and Romania (Man in´t Veld et al. 2015, Nechwatal et al. 2014), as well as in the USA (Reeser et al. 2015, Gitto et al. 2018). This is the first report of P. occultans in the Czech Republic. This pathogen likely poses another significant threat to boxwood cultivation in addition to the previously invaded Cydalima perspectalis and Calonectria pseudonaviculata.

Pseudoboletus silvaticus (Boletaceae, Basidiomycota), a new species from Mexico

The genus Pseudoboletus includes parasitic fungi and is well known in temperate zones. In this paper, we present the first Mexican collections of this genus. Two specimens were collected in a field expedition in a tropical montane cloud forest on the San Martín Tuxtla volcano, Veracruz, Mexico, resulting in a new species of Pseudoboletus, which grows on fruiting bodies of Scleroderma nitidum. Descriptions, illustrations, and photographs of P. silvaticus sp. nov. are provided. Also, its phylogenetic position in the Boletineae tree was inferred using nuclear 28S rDNA (28S) and translation elongation factor-1α (tef–1α) with Maximum Likelihood method and Bayesian Inference. Comparison between nuclear rDNA internal transcribed spacer region (ITS) sequences of P. parasiticus from North America and Europe and the new taxon were made. The original diagnosis of the genus Pseudoboletus is emended to include this new species.

First report of Calonectria ilicicola causing fruit rot on postharvest Prunus persica in Zhejiang Province, China.

Peach (Prunus persica) is an important economic tree fruit in China, with 15 million tons produced in 2020 (Xu et al. 2022). In September 2021, fruit rot on postharvest P. persica 'Yingqingtao' was observed in an orchard warehouse in Qixing district (120°41'E, 29°15'N), Zhejiang Province. Disease incidence was estimated at 25%, and yield loss was estimated at approximately 20% of the total yield. The naturally infected fruit had water-soaked, light brown lesions that fused, and produced a gray-white, dense mycelium (Fig. 1 A). The mycelia were transferred using a sterilized toothpick to potato dextrose agar (PDA) and cultured for 7 d. Macroconidia were used to produce five single-spore isolates, each from a different fruit. Six-day-old colonies grown on PDA at 26°C had light brown centers with gray-white edges; on the underside the centers were reddish brown and white towards the margin (Fig. 1 D). Isolate TGF2 was selected for further identification. Macroconidia were hyaline, straight, cylindrical, and one-to-three septae, 63.2 to 81.8 × 5.7 to 7.8 μm (mean = 73.9 ± 4.3 × 6.9 ± 0.5, n = 30) (Fig. 1 E). Chlamydospores were produced abundantly on PDA (Fig. 1 F), and measured 11.7 to 19.4 × 8.5 to 16.9 μm (n = 10). Perithecia were reddish orange, globose, and 329.9 to 417.1 μm in diameter on PDA (Fig. 1 G). Asci were hyaline and clavate, 61.2 to 91.8 × 14.4 to 20.7 μm (n = 10); ascospores were hyaline, slightly curved, 1- to 3-septate, mostly 1-septate, and 37.6 to 59.7 × 4.9 to 6.4 μm (mean = 49.9 ± 4.5 × 5.6 ± 0.4, n = 30) (Fig. 1 H-J). Morphological characteristics placed this organism within the Ca. kyotensis species complex (Liu et al. 2020). For molecular identification, the internal transcribed spacer (ITS: OP164807-OP164811), calmodulin (Cal: OP176049-OP176053), histone3 (His3: OP176054-OP176058), and translation elongation factor 1α (Tef1: OP176044-OP176048) genes were sequenced (Liu, et al., 2020). The twenty sequences were deposited in GenBank. A BLAST search of these sequences showed 99% identity with sequences of the ex-holotype Ca. ilicicola CMW 30998 (Liu et al. 2020). Bayes phylogenesis suggested that these strains and Ca. ilicicola CMW 30998 were clustered in the same clade (Bayesian posterior probability = 1) (Fig. 2). Integrating morphology and molecular data, these strains were identified as Ca. ilicicola. For pathogenicity tests, P. persica fruits were surface sterilized in 75% ethanol for 30 s and air-dried for 5 mins to allow the alcohol to volatilize. A conidial suspension (30 mL of 1 × 106 conidia/mL) of TGF2 was sprayed onto ten fruits, and ten fruits sprayed with sterilized water served as controls. The experiment was repeated three times. Fruits were kept on a mist bench at 26°C and 60% relative humidity. After 5 days, inoculated fruits showed necrotic lesions and a dense, gray-white mycelium, however, the control fruits showed no symptoms (Fig. 1 B, C). Ca. ilicicola was reisolated from lesions of inoculated fruits. Ca. ilicicola has been reported from Vaccinium sp., Glycine max, Medicago sativa (Farr and Rossman 2022; Kleczewski et al. 2019; Zhang et al. 2020). To our knowledge, this is the first report of Ca. ilicicola causing fruit rot of P. persica in China. In other research on Ca. ilicicola, we found that continuous light could inhibit its growth, suggesting a method to protect postharvest peaches.

Diaporthe species associated with the maritime grass Festuca rubra subsp. pruinosa.

Festuca rubra subsp. pruinosa is a perennial grass growing in sea cliffs where plants are highly exposed to salinity and marine winds, and often grow in rock fissures where soil is absent. Diaporthe species are one of the most abundant components of the root microbiome of this grass and several Diaporthe isolates have been found to produce beneficial effects in their host and other plant species of agronomic importance. In this study, 22 strains of Diaporthe isolated as endophytes from roots of Festuca rubra subsp. pruinosa were characterized by molecular, morphological, and biochemical analyses. Sequences of the nuclear ribosomal internal transcribed spacers (ITS), translation elongation factor 1-α (TEF1), beta-tubulin (TUB), histone-3 (HIS), and calmodulin (CAL) genes were analyzed to identify the isolates. A multi-locus phylogenetic analysis of the combined five gene regions led to the identification of two new species named Diaporthe atlantica and Diaporthe iberica. Diaporthe atlantica is the most abundant Diaporthe species in its host plant, and Diaporthe iberica was also isolated from Celtica gigantea, another grass species growing in semiarid inland habitats. An in vitro biochemical characterization showed that all cultures of D. atlantica produced indole-3-acetic acid and ammonium, and the strains of D. iberica produced indole 3-acetic acid, ammonium, siderophores, and cellulase. Diaporthe atlantica is closely related to D. sclerotioides, a pathogen of cucurbits, and caused a growth reduction when inoculated in cucumber, melon, and watermelon.

First report of leaf blight on Eucalyptus cloeziana caused by Coniella quercicola in China.

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.

First Report of Leaf Spot on Basella rubra Caused by Fusarium proliferatum in Malaysia.

Basella rubra (family Basellaceae), locally known as 'Remayong Merah', is the edible perennial vine served as leafy vegetable in Malaysia. In May 2021, B. rubra's leaves with circular to subcircular purple spots (ranging from 1-10 mm wide) were collected in Lido (5°56'44.6"N 116°04'46.5"E), Sabah province. The disease severity was about 60% with 20% disease incidence on fifty plants. As disease developed, the spots grew larger and necrosis were formed within the purple spots. Small pieces (5 x 5 mm) of five diseased spots were excised, and then surface sterilized based on Khoo et al. (2022b) before plating on water agar at 25°C. Once obtained the pure cultures from all diseased spots, they were incubated on potato dextrose agar at 25°C. After 7 days, white aerial mycelium with light violet pigmentation on lower side were observed on PDA. Then, the fungi were cultured on Carnation leaf agar (CLA) at 25°C and 12-h light/dark photoperiod for 10 days. Thin-walled slender and slightly curved macroconidia (n= 20) with 3 to 5 septa were ranged from 2.3 to 2.6 µm wide by 26.8 to 44.5 µm long in size. Oval microconidia (n= 20) with no septa were 2 to 2.2 µm wide by 9.5 to 15 µm long in size. Chlamydospores were absent. Monophialids with false head were observed. Isolate Lido and Lido02 were kept in the Laboratory of Genetics, Faculty of Science and Natural Resources, Universiti Malaysia Sabah for public request. Their genomic DNA were extracted from fresh mycelia of isolates based on Khoo et al. (2022a). EF1/EF2, RPB1-Fa/RPB1-G2R and RPB2-5f2/RPB2-7cr (Jiang et al. 2021) were used to amplify the translation elongation factor 1-α (TEF1) region, RNA polymerase largest subunit gene (RPB1) and RNA polymerase second largest subunit gene (RPB2) based on PCR condition in Khoo et al. (2022b). The isolate's sequences were deposited in GenBank as OM048109, OM634654 (TEF1), OM634655, OM634657 (RPB1) and OM634656, OM634658 (RPB2). They were 99 to 100% homology to TEF1 of isolate DPCT0102-2 (LC581453) (657/657 bp), RPB1 of strain ZJ05 (MT560605) (1558/1558 bp) and RPB2 of isolate GR_FP248 (MT305154) (1867/1869 bp) sequences. These sequences were polyphasic identified at the Fusarium MLST (https://fusarium.mycobank.org/), and were more than 99% similarity to Gibberella fujikuroi species complex (NRRL 25200). Gibberella fujikuroi and Fusarium fujikuroi are synonymous with Fusarium proliferatum (Leslie and Summerell 2006). The pathogen was identified as F. proliferatum based on morphological characterization, molecular data and phylogenetic analysis. Two non-wounded leaves of three one-month-old B. rubra seedlings were inoculated with mycelium plug (10 x 10 mm). Additional three B. rubra seedlings received sterile PDA agar plug (10 x 10 mm) to serve as controls. They were incubated in a glasshouse at room temperature 25°C with a relative humidity of 80 to 90%. After 8 days of inoculation, all inoculated leaves exhibited the symptoms as observed in the field, while the controls showed no symptoms, thus confirming the Koch's postulates. The experiment was repeated two more times. The reisolated pathogens were identified as F. proliferatum via PDA macroscopically, CLA microscopically and PCR amplification. F. proliferatum was reported previously causing leaf spot disease on Cymbidium orchids (Wang et al. 2018), tobacco (Li et al. 2017) and tomato (Gao et al. 2017). To our knowledge, this is the first report of F. proliferatum causing leaf spot on B. rubra in Malaysia. Infections of leaves reduce plant vigor and marketability. The identification of leaf spot caused by F. proliferatun will enable plant health authorities and farmers to identify practices to minimize disease on this important crop.

First Report of Diaporthe phoenicicola causing leaf spot on blueberry (Vaccinium virgatum) in China.

Blueberry (Vaccinium virgatum), a member of the Ericaceae family, is an increasingly important crop in China because of its abundant nutritional benefits and economic value (Kuzmanović et al. 2019). In October 2021, leaf spots were detected on 'Rabbiteye' blueberry at the Agricultural Science and Technology Park of Jiangxi Agricultural University in Nanchang, China (28°45'51"N, 115°50'52"E), which caused severe defoliation of the crop and fruit yield losses of 25% (Figure 1A). Disease surveys were conducted at that time; the results showed that disease incidence was 75.5%, observed in 151 of the 200 accessions sampled, and this disease had not been found at other cultivation fields in Nanchang. Lesions with taupe to dark brown margins were irregularly shaped and associated with leaf margins. Spots coalesced to form larger lesions, with black pycnidia present in more mature lesions. To identify the causal agent, 10 small pieces (5 mm2) of leaf tissue excised from the lesion margins were surface sterilized in 75% ethanol solution for 30 s and 0.1% mercuric chloride solution for 2 min, rinsed three times with sterile distilled water, then placed on potato dextrose agar (PDA) at 25°C for 5 to 7 days in darkness. Five fungal isolates showing similar morphological characteristics were obtained as pure cultures by single-spore isolation. All fungal colonies on PDA were floccose, dense, and white (Figure 1B.C). Black pycnidia developed on PDA at 25°C under a 12/12 h light/dark cycle for 30 days. Alpha conidia were 6.17 to 8.53 × 1.64 to 3.20 µm (average 6.94 × 2.52 µm, n = 100), aseptate, hyaline, fusiform to ellipsoidal, often biguttulate. Beta conidia were 15.26 to 25.41 × 0.92 to 1.40 µm (average 20.14 × 1.27 µm, n = 30), aseptate, hyaline, linear to hamate (Figure 1D). Based on morphological characteristics, the fungal isolates were suspected to be Diaporthe spp. (Gomes et al. 2013). To further confirm the identity of this putative pathogen, two representative isolates (LGM1 and LGM2) were selected for molecular identification. The internal transcribed spacer region (ITS), translation elongation factor 1α (EF1-α), histone H3 (HIS), calmodulin (CAL), and β-tubulin (TUB2) genes were amplified from gDNA and sequenced using primers ITS1/ITS4 (Peever et al. 2004), EF1-728F/EF1-986R and CAL228F/CAL737R (Carbone et al. 1999), CYLH3F/H3-1b (Crous et al. 2004), Bt2a/Bt2b (Glass and Donaldson 1995), respectively. GenBank accession numbers of isolate LGM1 and LGM2 were OM778771 to 72 for the ITS region, OM868228 to 29 for EF1-α, OM837771 to 72 for TUB2, ON206971 to 72 for CAL, ON206973 to 74 for HIS. BLAST results showed that the ITS, EF1-α, TUB2, HIS, and CAL sequences showed 99% (538/545 bp), 100% (322/322 bp), 99% (480/484 bp), 99% (459/460 bp), 99% (430/433 bp) identity, respectively, with those of Diaporthe phoenicicola (GenBank accession no. MW504735, MW514099, MW514142, MW514067, MT409304). Two maximum likelihood phylogenetic trees were built based on the sequences of ITS, EF1-α, HIS, CAL, and TUB2 by using MEGA 5. The two isolates LGM1 and LGM2 clustered with D. phoenicicola (Figure 2 and 3). The fungus was identified as D. phoenicicola by combining morphological and molecular characteristics. To evaluate the pathogenicity, three healthy young potted V. virgatum plants were spray inoculated with a conidial suspension of 106 conidia/ml. Another set of three plants that were sprayed with sterilized distilled water served as the controls. The experiment was repeated three times, and all plants were maintained in a climate box (12 h light/dark) at 25°C with 80% relative humidity. Five days after inoculation, no symptoms were observed on control plants (Figure 1F), and all inoculated plants showed symptoms (brown flecks) similar to those observed in the field (Figure 1E). The fungus was reisolated from the infected tissues and confirmed as D. phoenicicola by morphological and molecular identification, and could not be isolated from the controls, fulfilling Koch's postulates. To our knowledge, this is the first report of D. phoenicicola causing leaf spot on blueberry in China. The discovery of this new disease and the identification of the pathogen will provide useful information for developing specific control measures and potential sources for resistance to this disease caused by D. phoenicicola.

Leaf vein disease in Nelumbo nucifera caused by Nigrospora pyriformis infection in China.

Nelumbo nucifera (Nymphaeaceae) has both ornamental and nutritional uses in China. In July 2021, a novel disease was observed on plants in the White Lotus Science and Technology Expo Park in Guangchang county, Fuzhou city, Jiangxi province (26.79° N, 116.31° E). Infection was visible as nearly round black spots on the leaves in the early stages of infection. At later stages, the spots spread along the veins forming reticular necrosis until the entire leaf was infected, and the disease caused approximately 20% of leaves to die. To identify the pathogenic organism, 5×5 mm samples were taken from affected tissue adjoining healthy tissue, sterilized in 75% ethanol for 30 s, and immersed in 0.1% mercury chloride for a further 30 s, before washing in sterile water and transfer to potato glucose agar (PDA) plates. After culturing at 28℃±1℃ for five days, white regularly shaped round colonies were visible that after 10 days turned black with fluffy hyphae. The individual conidia produced by the conidiophore were initially a light-brown color which changed to black as the pigment accumulated; conidia were globose to subglobose, 17.10 (14.77-21.66) ×14.70 (12.08-16.93) μm in size (n=50), with glossy even surfaces lacking in septa, suggestive of Nigrospora. To verify this, PCR amplification was conducted using ITS1/ITS4 primers for the 5.8S rRNA gene. The primers EF1-728F and EF-2 were used for amplifying translation elongation factor 1α (O'Donnell et al. 1998, Carbone and Kohn 1999), and Bt-2a and Bt-2b (Glass and Donaldson 1995) for amplifying β-tubulin. The sequences were found to be 99% to 100% identical to those of Nigrospora pyriformis in GenBank (accession numbers NR153469.1, KY019290.1, and KY019457.1, respectively). The sequences were uploaded to GenBank (accession numbers OK605048, OL362197, and OK662966), with lengths of 522 bp, 475 bp, and 410 bp, respectively. A maximum-likelihood phylogenetic tree was created in MEGA5. Pathogenicity was tested by four isolates hyphal inoculation and conducted in an experimental field of the White Lotus Science and Technology Expo Park. Five-millimeter discs were taken from infected and uninfected PDA plates and inoculated into five 1-month-old healthe lotus leaves using inoculations, three with the pathogen and an uninfected sample as the control every leaf. The discs were covered with moisturized sterile cotton and fixed with transparent tape. The wounds were moistened with sterile water and sealed with adhesive tape. After 14 days, spots were visible at the infected sites while the control sites showed no symptoms. The same pathogen was recovered from the infected leaves, fulfilling Koch's requirements. Leaf spot diseases resulting from N. pyriformis infection or infection with other Nigrospora species include infection of Chenopodium album by N. pyriformis (Chen et al. 2020), Eclipta prostrata by N. sphaerica (Qiu et al. 2022), Nicotiana tabacum by N. oryzae (Wang et al. 2022), and Oxalis corymbosa by N. hainanensis (Zheng et al. 2021). This investigation appears to be the first identification of Nelumbo nucifera vein disease resulting from Nigrospora pyriformis infection. The present results are useful for the management and avoidance of the disease caused by Nigrospora pyriformis.

Identification and First Report of Leaf Blight Caused by Phytophthora palmivora on Chinese Water Bamboo in China.

Chinese water bamboo (Dracaena sanderiana) is a popular houseplant due to its ability to survive in various indoor conditions. In October 2020, pronounced leaf blight symptoms with approximately 50% disease incidence were observed on water bamboo in the 35-ha field of Wenchang county (19°50'45'' N, 110°21'38'' E), Hainan, China. The diseased leaves showed pale green with yellowish lesions, dried and shrivelled tips. As the disease progressed, upward-spreading necrosis led to stem weakness, and then the plants wilted and died within a few weeks (Fig. 1A,B). Ten symptomatic leaves were collected, and leaf pieces (5 mm × 5 mm) from the edge of the lesion were cut, disinfected for 30 s using 75% ethanol, and rinsed five times in d.d. H2O, and placed on V8 media amended with 10 mg/L pimaricin, 150 mg/L ampicillin, and 16 mg/L rifampicin (Guo et al., 2012). Eight isolates of Phytophthora recovered from 10 leaves were characterized morphologically. Colony morphology showed slightly radiate to stellate patterns, with cottony aerial mycelium (Fig.1D). Hyphae were smooth and uniform, branching at nearly right angles into stout (Fig.1E). Mycelial swellings were observed in plate culture (Fig.1F). Chlamydospores were abundant and spherical (Fig.1G). Sporangia were produced externally through sympodial development of the sporangiophore immediately below a sporangium (Fig.1H, I). The sporangia were 29.3-63.9 µm (mean, 48.5 µm) in length and 19.3-46.8 µm (mean, 33.5 µm, n=50) in width. No sexual organs were observed in the 8 isolates. The morphological characteristics of the isolates were consistent with the description of P. palmivora (Erwin and Ribeiro, 1996). The internal transcribed spacer (ITS) region of rDNA, the translation elongation factor 1α gene (TEF1), and β-Tubulin gene (TUB) sequences of the 8 isolates were amplified using the primers ITS1/ITS4 (Cooke et al., 2000), ELONGF1/ELONGR1 and TUBUF2/TUBUR1 (Kroon, et al., 2004), respectively, followed by sequencing analysis. The sequences of representative isolate HK-5 for the ITS, TEF1 and TUB were deposited in GenBank with the accession numbers of OK349485, OK349488 and OK349487. BLAST results showed that the ITS (MG865559, identity=735/735; 100%), TEF1(MH359047, identity=682/684; 99.7%) and TUB (MH493992, identity=467/468; 99.8%) sequences were all highly similar with a sequence of P. palmivora strain CPHST BL105. Phylogenetic analysis using the combined ITS, TEF1, and TUB sequences showed that the isolate HK-5 were grouped with the P. palmivora with good bootstrap support (Fig.2). Based on morphological and molecular identification, the pathogens were identified as P. palmivora. The pathogenicity tests showed that all whole plants (1-year-old) sprayed with the 5 mL of zoospore suspension (1×106 zoospores mL-1) initially presented with discoloured spots on their leaves after 4 days of inoculation, and the symptoms gradually progressed from spots to leaf blight (Fig. 1C). Each isolate was applied onto 10 plants and control plants were sprayed with d.d.H2O. The results of the pathogenicity test exhibited typical symptoms as observed in the field. No significant differences of virulence were observed among the 8 isolates, and the control plants remained symptomless. P. palmivora was re-isolated from the leaves of inoculated plants. This is the first report of P. palmivora on water bamboo in China, and appropriate measures must be undertaken to control this agent in this region.

Fungal species characterization associated with vanilla root rot in the Totonacapan, Veracruz, Mexico

Vanilla (Vanilla planifolia) is a crop of great biocultural importance in the Totonacapan region in Mexico; however, producers face various phytosanitary problems that decrease production. The main one being crop loss caused by the fungus, Fusarium oxysporum f. sp. vanillae, a pathogen that causes a disease whose symptomatology is root rot that in severe cases causes the death of plants. Therefore, the study of the pathogen is crucial, due to the scarcity of genetic variability of vanilla, a condition that makes the crops equally susceptible to the pathogen. The present work aimed to generate and molecularly and pathogenically characterize a fu-ngal isolates collection obtained from V. planifolia with root rot. Roots with rot were collected in six municipalities of the Totonacapan region, for fungal isolation, a semiselective culture medium was used, later, the molecular characterization was carried out through the amplification and sequencing of the partial region of the translational elongation factor 1α gene (TEF1). The pathogenic characterization was carried out on vanilla leaves in a humid chamber, inoculating medium discs with mycelium, and making observations for 18 days every 48 h. As results, 94 strains were obtained, of which 81 belong to the genus Fusarium (42 F. oxysporum strains), eight to Colletotrichum, three to Trichoderma and two it was not possible to identify them, only 23 Fusarium oxysporum strains showed to be pathogenic on vanilla leaves with different degrees of virulence. The results of the molecular and pathogenic characterization will be crucial for further studies of comparative genomics of Fusarium oxysporum.

Fungal Pathogens Associated with Crown and Root Rot in Wheat-Growing Areas of Northern Kyrgyzstan.

Fungal species associated with crown and root rot diseases in wheat have been extensively studied in many parts of the world. However, no reports on the relative importance and distribution of pathogens associated with wheat crown and root rot in Kyrgyzstan have been published. Hence, fungal species associated with wheat crown/root rot were surveyed in three main wheat production regions in northern Kyrgyzstan. Fungal species were isolated on 1/5 strength potato-dextrose agar amended with streptomycin (0.1 g/L) and chloramphenicol (0.05 g/L). A total of 598 fungal isolates from symptomatic tissues were identified using morphological features of the cultures and conidia, as well as sequence analysis of the nuclear ribosomal internal transcribed spacer (ITS) region, the translation elongation factor 1α (TEF1), and the RNA polymerase II beta subunit (RPB2) genes. The percentage of fields from which each fungus was isolated and their relative percentage isolation levels were determined. Bipolaris sorokiniana, the causal agent of common root rot, was the most prevalent pathogenic species isolated, being isolated from 86.67% of the fields surveyed at a frequency of isolation of 40.64%. Fusarium spp. accounted for 53.01% of all isolates and consisted of 12 different species. The most common Fusarium species identified was Fusarium acuminatum, which was isolated from 70% of the sites surveyed with an isolation frequency of 21.57%, followed by Fusarium culmorum, Fusarium nygamai, Fusarium oxysporum, and Fusarium equiseti, all of which had a field incidence of more than 23%. Inoculation tests with 44 isolates representing 17 species on the susceptible Triticum aestivum cv. Seri 82 revealed that Fusarium pseudograminearum and F. culmorum isolates were equally the most virulent pathogens. The widespread distribution of moderately virulent B. sorokiniana appears to be a serious threat to wheat culture, limiting yield and quality. With the exception of F. culmorum, the remaining Fusarium species did not pose a significant threat to wheat production in the surveyed areas because common species, such as F. acuminatum, F. nygamai, F. oxysporum, and F. equiseti, were non-pathogenic but infrequent species, such as Fusarium redolens, Fusarium algeriense, and F. pseudograminearum, were highly or moderately virulent. Curvularia inaequalis, which was found in three different fields, was mildly virulent. The remaining Fusarium species, Fusarium solani, Fusarium proliferatum, Fusarium burgessii, and Fusarium tricinctum, as well as Microdochium bolleyi, Microdochium nivale, and Macrophomina phaseolina, were non-pathogenic and considered to be secondary colonizers. The implications of these findings are discussed.

Isolation, identification, biological characteristics, and antifungal efficacy of sodium bicarbonate combined with natamycin on Aspergillus niger from Shengzhou nane (Prunus salicina var. taoxingli) fruit.

The fungi causing fruit rot were isolated from symptomatic Shengzhou nane (Prunus salicina var. taoxingli) fruit and were identified as Aspergillus niger by biological characteristics and molecular analysis of the internal transcribed spacer region (rDNA-ITS) and translation elongation factor-1α (TEF-1α) sequences. Optimal growth conditions for A. niger were 30°C, pH 5.0-6.0, and fructose and peptone as carbon and nitrogen sources. The effects of sodium bicarbonate (SBC), natamycin (NT), and combined treatments on A. niger inhibition were investigated. Treatment with 4.0 g/L sodium bicarbonate (SBC) + 5.0 mg/L natamycin (NT) inhibited mycelial growth and spore germination as completely as 12.0 mg/L SBC or 25.0 mg/L NT. SBC and NT treatments disrupted the structural integrity of cell and mitochondria membranes and decreased enzyme activities involved in the tricarboxylic acid (TCA) cycle, mitochondrial membrane potential (MMP), ATP production in mitochondria, and ergosterol content in the plasma membrane, thus leading to the inhibition of A. niger growth. Moreover, experimental results in vivo showed that the rot lesion diameter and decay rate of Shengzhou nane fruit treated with SBC and NT were significantly reduced compared with the control. The results suggest that the combination treatment of SBC and NT could be an alternative to synthetic fungicides for controlling postharvest Shengzhou nane decay caused by A. niger.

Diagnosis and Control of Brown Leaf Spot of Kiwi (Actinidia deliciosa) Using Biochar-Zinc Oxide Nanocomposite (MB-ZnO) as a Non-Toxic Bio-Fungicides

Kiwi is one of the best natural sources of vitamin C and has wide applications. During October–November 2021, small brown spots were examined on the Kiwi leaves. The diseased leaf samples were collected and placed on potato dextrose agar nutrient media for diagnosis. Morphological, anatomical, and molecular studies revealed this disease-causing agent to be Rhizopus oryzae. Molecular characterizations of the isolated pathogen were performed by using actin translation elongation factor (EF-1α) and ribosomal deoxyribose nucleotide inter transcribed sequence (rDNA ITS ITS1/ITS4) and elongation factors (EFl-F/EFl-R) primers. A BLAST study of the resultant ITS1/ITS4 sequence showed > 99% resemblance with R. oryzae (MT603964.1), while the EF-1α sequence revealed 100% similarity with translation elongation factor-1α gene of R. oryzae (MK510718.1). The obtained ITS1/ITS4 sequence was submitted to NCBI (MW603842.1). Koch’s postulates established the pathogenicity of isolated R. oryzae and proved it to be the brown spot pathogen of Kiwi. For the environmentally-friendly management of Kiwi leaf spot, maize biochar-Zinc Oxide (MB-ZnO) nanocomposite was used. The prepared nanocomposite was characterized by Fourier transform infrared (FTIR) spectroscopy, thermo gravitational analysis (TGA), X-ray diffraction (XRD) analysis, scanning electron microscopy (SEM), and energy dispersive X-ray (EDX) analysis. After successful preparation, MB-ZnO was assessed for its possible antifungal potential against R. oryzae. MB-ZnO displayed substantial growth inhibition, and the highest growth inhibition (79%) was observed at a 19 mg/mL dose rate of nanoparticles. These excellent findings propose that Ball-milled synthesis is a fast, economical, and environmentally friendly method for nanocomposite in the near future. The nanocomposite is used as a nominal substitute for chemical fungicides.

Expanding the spectrum of EEF1D neurodevelopmental disorders: Biallelic variants in the guanine exchange domain.

Protein translation is an essential cellular process and dysfunctional protein translation causes various neurodevelopmental disorders. The eukaryotic translation elongation factor 1A (eEF1A) delivers aminoacyl-tRNA to the ribosome, while the eEF1B complex acts as a guanine exchange factor (GEF) of GTP for GDP indirectly catalyzing the release of eEF1A from the ribosome. The gene EEF1D encodes the eEF1Bδ subunit of the eEF1B complex. EEF1D is alternatively spliced giving rise to one long and three short isoforms. Two different homozygous, truncating variants in EEF1D had been associated with severe intellectual disability and microcephaly in two families. The published variants only affect the long isoform of EEF1D that acts as a transcription factor of heat shock element proteins. By exome sequencing, we identified two different homozygous variants in EEF1D in two families with severe developmental delay, severe microcephaly, spasticity, and failure to thrive with optic atrophy, poor feeding, and recurrent aspiration pneumonia. The EEF1D variants reported in this study are localized in the C-terminal GEF domain, suggesting that a disturbed protein translation machinery might contribute to the neurodevelopmental phenotype. Pathogenic variants localized in both the alternatively spliced domain or the GEF domain of EEF1D cause a severe neurodevelopmental disorder with microcephaly and spasticity.