Translation Elongation Research Articles

Armillaria Species Causing Kiwifruit Vine Decline and Root Rot in Northeastern Türkiye: A Growing Concern for Kiwifruit Health

ABSTRACTKiwifruit (Actinidia deliciosa) production in northeastern Türkiye is facing an emerging threat from Armillaria root rot. Surveys conducted in August 2021 and 2022 revealed that 10.5%–17.5% of the 200–500 vines within each of 35 kiwifruit orchards in the Perşembe, Gülyalı and Altınordu districts of Ordu province exhibited symptoms of Armillaria root rot, including complete defoliation. Diseased plants exhibited diagnostic signs on their root collars and woody roots, including white mycelial fans, extensive wood rot and rhizomorphs. A single Armillaria isolate was obtained from each of the 35 symptomatic orchards. Molecular identification, employing DNA sequencing of the large subunit (LSU) region of rDNA, the translation elongation factor subunit 1‐alpha (TEF1) gene and the second largest RNA polymerase II B‐subunit (RPB2) gene, identified 20 isolates as Armillaria gallica and 15 as A. mellea. Phylogenetic analyses, based on TEF1 alignments, further supported the species identification with a 100% bootstrap value. To examine genetic diversity, start codon targeted (SCoT) marker 13 was used, successfully differentiating the two Armillaria species and revealing three distinct clades. Clades I and II comprised 15 and 5 isolates of A. gallica, respectively, while Clade III contained all 15 A. mellea isolates. Pathogenicity testing on kiwifruit seedlings (cv. Hayward) revealed the ability of both A. gallica and A. mellea isolates to induce disease. Rhizomorph‐producing isolates in kiwifruit seedlings displayed high virulence, inducing severe leaf necrosis and seedling mortality, while isolates lacking these structures were non‐pathogenic. This study identifies the first global instance of A. gallica causing kiwifruit root rot. This finding, along with the identification of A. mellea as a causal agent in the country, highlights the emerging threat of Armillaria root rot to kiwifruit production in the region.

First Report of Root Rot Caused by Fusarium avenaceum and Fusarium flocciferum on Astragalus membranaceus var. mongholicus in China

ABSTRACTAstragalus membranaceus var. mongholicus (Mongolian milkvetch) is one of the most important traditional Chinese medicinal plants. In July 2021, root rot of A. membranaceus var. mongholicus was found in a field in Tongren City, Huangnan Tibetan Autonomous Prefecture, Qinghai Province, China. The aboveground part of the plants exhibited yellowing, wilting and defoliation in severe cases. Seven fungal isolates were obtained, and their pathogenicity, morphology and phylogeny were analysed. A pathogenicity test was performed using the scratch and root irrigation methods, and the results showed that all seven isolates caused root rot on inoculated plants. Based on morphological characteristics and phylogenetic analysis of multi‐locus sequences (ITS, translation elongation factor [TEF‐1α], and RNA polymerase II beta subunit [RPB2] genes), two isolates were identified as Fusarium avenaceum, and five isolates were identified as F. flocciferum. To the best of our knowledge, this is the first report of F. avenaceum and F. flocciferum causing root rot of A. membranaceus var. mongholicus in China.

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

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

First Report of Nigrospora oryzae Causing Leaf Spot on Peanut (Arachis hypogaea L.) in the Republic of Korea.

Peanut (Arachis hypogaea L.) is one of the most profitable upland crops, yielding 10,711 tonnes in an area of 4,062 ha in the Republic of Korea (Ministry of Agriculture, Food and Rural Affairs 2023). In September 2023, dark gray spots surrounded by yellow halos were observed on the peanut leaves over an area of 880 m2 at the National Institute of Crop Science (35°50'31.4"N 127°02'41.0"E), with a disease incidence up to 80%. Early symptoms appeared as small, brown, circular or irregular spots that enlarged and were surrounded by chlorotic halos. Leaf cuttings (5 mm x 5 mm) from five symptomatic plants were surface-sterilized with 70% EtOH for 1 min, followed by 1% NaClO for 1 min, and rinsed 3 times with sterile water. The pieces were placed on Potato Dextrose Agar (PDA) and incubated at 25 °C in the dark for 3 days. Three isolates obtained by single-spore isolation were designated as F23025, F23026, and F23027. Two isolates, F23025 and F23026 were deposited in the Korean Agricultural Culture Collection (https://genebank.rda.go.kr) under the accession numbers 410722 and 410723. Fungal colonies were initially white and turned sooty gray after 5 days. Conidia were unicellular, brown to black, and spherical or sub-spherical with 6.8 μm to 14.3 μm (mean = 11.1 μm ± 1.8, n = 50). The morphology of the three isolates was identical and showed the same characteristics as Nigrospora oryzae (Ellis 1971; Hudson 1963). For molecular identification, the Internal Transcribed Spacer (ITS) region (GenBank accession PP388306 and PP574448), beta tubulin (PP397027 and PP580108), and translation elongation factor 1- ɑ (PP397028 and PP580109) of isolates F23025 and F23026 were amplified and sequenced with primers of ITS5/ITS4, Bt2a/Bt2b, EF1-727F/EF2, respectively and showed high identity of 99.62% (530 bp/532 bp), 100% (384/384), and 99.79% (475/476) with N. oryzae strain LC2693 (GenBank accessions KX985994, KY019471, and KY019299, respectively). Multilocus sequence analysis showed isolates F23025 and F23026 were on the same clade with N. oryzae strain LC2693. To determine the pathogenicity to peanut, a conidial suspension (1 x 106 conidia/mL) was sprayed onto leaves of five 3-week-old plants 'Sewon' grown in pots, while sterile distilled water was sprayed onto two plants used as negative control. Sprayed plants were placed in a dew chamber at 25℃ for two days and grown in a growth chamber at 25℃ and 80% of relative humidity with a 16L:8D cycle. Two weeks later, dark spots with chlorotic halos appeared only on leaves sprayed with conidia, and no symptoms on leaves sprayed with sterile distilled water. The pathogenicity test was repeated three times, and each time the pathogen was re-isolated and identified by ITS sequence, thus fulfilling Koch's postulates. Nigrospora species are cosmopolitan, and some species have a wide host range as plant pathogens. Recently, two species of the genus Nigrospora, N. sphaerica and N. aurantiaca, were reported to cause peanut leaf blight in China (Liu et al. 2020; He et al. 2023). To the best of our knowledge, this is the first report of N. oryzae causing leaf spot to A. hypogaea L. in the Republic of Korea. As identifying new pathogens and registering fungicides to control them are important for the continued cultivation of peanut, this report will help in that endeavor.

First Report of Lasiodiplodia theobromae Causing Fruit Crown Rot on Banana in Ecuador.

Post-harvest diseases like fruit crown rot (CR) on bananas (Musa spp.) worldwide are mainly attributed to Colletotrichum gloeosporioides (Berk. & Curt.) von Arx and Lasiodiplodia theobromae (Pat.) Griff. & Maubl (Sangeetha et al., 2012; Riera et al., 2019). In April 2019, at a banana farm (cultivar Williams) located in El Oro province (location at 79° 54' 05" W; 03° 17' 16" S), thirty hands were randomly collected from the postharvest process and further placed in a humid chamber at 20 ºC until signs of the disease progressed and became more evident (from 3 days to 20 days). Ten hands presented initial symptoms related to CR during the postharvest process, which included crown or peduncle rot with mycelial development on the crown's surface, leading to the blackening of tissues at the site of the wound left when the cluster was cut. Crown fruit fragments (~0.5 cm) from the edge of healthy tissue and diseased tissue underwent a series of disinfection steps, initially in ethanol (70%) for 1 min, followed by sodium hypochlorite (1%) for 1 min, rinsed three times with sterile distilled water, and dried on sterile filter paper for 10 min. The fragments were placed onto Potato dextrose agar (PDA) + chloramphenicol (100 mg L-1) and incubated at 25°C in darkness for five days. Five isolates with different colony morphologies were obtained. An initial screen of the pathogenicity of all isolates showed that only one isolate showed disease activity in banana crowns. This isolate, C1, showed grayish-white aerial mycelium in culture as described above and, after ten days, became black. We did a full pathogenicity test with C1 using ten individual banana fruits (cv. Williams Cavendish). Briefly, one disc (Ø of 5 mm) of the fungus with agar was placed on the acropetal part of the banana fruit (on the peel) and another piece in the crown without wounding. Inoculated fruit were in a humid chamber at 20 °C for 20 days. Uninoculated fruits constituted the control. Isolate C1 caused 100% of the fruit and crowns to rot, with symptoms similar to those initially observed from fruit collected at the postharvest process (Fig. S1d). The fungus was re-isolated from symptomatic tissue, and its identity was confirmed through morphological characteristics consistent with Lasiodiplodia sp. Matured conidia of all mono hyphal strains (Fig. S1b) appeared dark brown with a single septum, having an ovate shape, and displayed longitudinal striations along their thickened walls (Fig. S1c). The dimensions of the mature conidia ranged from 16.02 - 26.85 x 11.09 - 16.74 µm (n = 60). Morphological characteristics showed similarity to Lasiodiplodia sp. (Alves et al., 2008). Microscopic observations were further confirmed by sequencing three loci: the internal transcribed spacer (ITS), β-tubulin, and partial translation elongation factor-1α (TEF-1α). Fungal genomic DNA from the C1 isolate was PCR amplified using ITS5/ITS4, EF1-728F/986R, and Bt2A/Bt2B primers, respectively, according to Glass & Donaldson (1995) and Bautista-Cruz et al. (2019). The resulting amplicons were sequenced, and those sequences were deposited in GenBank with the accession numbers ITS: PP532861, TEF-1α: PP551938, and β-tubulin: PP537587. Sequence alignment was conducted using ClustalW under the MEGA 11.0 software package (Tamura et al., 2021). Subsequently, phylogenetic analysis was performed using Bayesian inference using the BEAST v1.8.4 program (Drummond & Rambaut, 2007). The concatenated sequence of the isolate revealed clustering to the Lasiodiplodia theobromae clade, confirming its identity. To our knowledge, this is the first report of this pathogen causing CR on banana fruit in Ecuador. Based on the report of CR in the country, banana exporters and the Ecuadorian government should consider developing disease management methods that include the cultivation, shipping, ripening, and storage processes of the fruit.

EEF1α2 is required for actin cytoskeleton homeostasis in the aging muscle.

The translation elongation factor eEF1α (eukaryotic elongation factor 1α) mediates mRNA translation by delivering aminoacyl-tRNAs to ribosomes. eEF1α also has other reported roles, including the regulation of actin dynamics. However, these distinct roles of eEF1α are often challenging to uncouple and remain poorly understood in aging metazoan tissues. The genomes of mammals and Drosophila encode two eEF1α paralogs, with eEF1α1 expressed ubiquitously and eEF1α2 expression more limited to neurons and muscle cells. Here, we report that eEF1α2 plays a unique role in maintaining myofibril homeostasis during aging in Drosophila. Specifically, we generated an eEF1α2 null allele, which was viable and showed two distinct muscle phenotypes. In young flies, the mutants had thinner myofibrils in indirect flight muscles that could be rescued by expressing eEF1α1. With aging, the muscles of the mutant flies began showing abnormal distribution of actin and myosin in muscles, but without a change in actin and myosin protein levels. This age-related phenotype could not be rescued by eEF1α1 overexpression. These findings support an unconventional role of Drosophila eEF1α2 in age-related homeostasis of muscle myofibers.

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

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

First Report of Seed Decay Caused by Diaporthe ueckeri on soybean in Brazil.

A survey of Diaporthe/Phomopsis Complex (DPC) species was carried out on 479 asymptomatic soybean (Glycine max (L.) Merrill) seed samples collected from commercial soybean fields in the states of Santa Catarina (20 counties) and Rio Grande do Sul (41 counties), in the 2020/21 (n=186), 2021/22 (n=138) and 2022/23 (n=155) seasons from 120 cultivars. The seeds were provided by seed producers who collected according to the sampling standard of the Ministry of Agriculture, Livestock and Food Supply. From each sample received, 200 symptomless seeds were randomly sorted out. The seeds were surface disinfected by immersion in a sodium hypochlorite solution (1%) for two minutes and placed on Potato Dextrose Agar (PDA). The plates were incubated for 7 days at 23°C with a photoperiod of 12-h. The average prevalence of 73.7% of DPC-infected seeds. Colonies were isolated by transferring mycelial tips to PDA and incubating for 14 days at 25ºC in a 12-h photoperiod. One colony (isolate MEMR0500) had morphological characteristics similar to those reported in Lopez-Cardona (2021). This isolate had a floccose, dense colony ranging from grayish beige to brown with greenish regions and black globose pycnidia (3 to 4 pycnidia/cm²). Alpha-conidia, 5.1 to 7.0 µm x 1.5 to 2.8 µm, were observed after 30 days and were hyaline, aseptate and fusiform (Figure S1). No beta-conidia were observed. Soybean plants of cultivars BMX Cromo IPRO, BMX Zeus IPRO, BRS 5804 RR, FPS 1867 IPRO and NEO 750 IPRO were tested for pathogenicity using the toothpick inoculation method (Siviero and Menten 1995). Non-colonized toothpicks served as a negative control. Plants were incubated for four days at 25°C and 90% relative humidity. Elongated 1.0 to 2.5 cm x 0.5 to 0.9 cm lesions gray-brown/reddish-brown with a depressed center were observed in all inoculated cultivars. The fungus was reisolated and the characteristics of the colonies were identical to those previously isolated. For molecular characterization, DNA was extracted from the mycelia using the CTAB method (Doyle and Doyle 1990). End-point PCR was performed using GoTaq® Flexi DNA Polymerase (Promega, USA) and primer pairs, ITS-4F/ITS-5, T2/Bt2b and EF1-728F/EF1-986R to amplify the internal transcribed spacer (ITS) (Costamilan et al. 2008), β-tubulin (TUB2) (Glass and Donaldson 1995), and translation elongation factor 1-α (TEF1) (Carbone and Kohn 1999) genes, respectively. The amplified fragments were sequenced and submitted to blast search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) with the sequences available in GenBank. The fragment from ITS (accession number OR912979) showed 99.8% (549/582 bp) identity with Diaporthe ueckeri Udayanga & Castl. [as 'ueckerae'] [syn. D. miriciae R.G. Shivas, S.M. Thomps. & Y.P. Tan] isolate FAU656 (Ac. N. KJ590726). The sequence of TEF (Ac. N. PP372869) showed 99.7% (339/355 bp) identity with D. ueckeri FAU656 (Ac. N. KJ590747), and of TUB (Ac. N. PP372870) showed 98.9% (436/536 bp) identity with D. ueckeri FAU656 (Ac. N. KJ610881). A phylogenetic tree with amplified sequences of each gene and the corresponding representative sequences from the DPC was constructed in MEGA X (Kumar et al. 2018). The MEMR0500 isolate was clustered only with the D. ueckeri clade, confirming the identity of the fungus (Figure S2). In Brazil, this is the first report of the association of this pathogen with soybean seeds. In other countries, this pathogen has been identified as the causal agent of stem canker (Mena et al. 2020; Lopez-Cardona et al. 2021). Further research is needed to analyze the risk of this seed-associated pathogen.

First report of Fusarium oxysporum f. sp. fragariae race 1 causing Fusarium wilt of strawberry (Fragaria × ananassa) in the Alentejo region of Portugal.

In the summer of 2023, within the Alentejo region (Portugal), a new occurrence of a plant disease of strawberry (Fragaria × ananassa) cv. 'Monterey' was observed in a Spring commercial planting. Symptoms consisted of foliar wilting, drying of older leaves, deformed and highly chlorotic leaflets, crown discoloration, plant stunting, and in some cases death. Several outbreak foci, covering nearly half a hectare, were observed within the affected farm, with almost 80% of the plants showing symptoms. Four samples,of 6 plants, were collected from 4 locations within the field. Petiole sections (1 cm) were rinsed with 0.1% Tween 20, submersed in 70% EtOH for 20 s followed by 60 s in 1% NaOCL, and then placed on Komada's medium (Komada, 1975). After incubation at room temperature in the dark for a week, white-colored fluffy mycelia grew profusely from the petioles of all samples. Colony morphology and non-septate, ellipsoidal microconidia (5.7-12.4×2.5-4.3 μm) borne on monophialides, exhibited a resemblance to Fusarium oxysporum (Leslie and Summerell, 2006). Ten single strains were obtained from different plants by single hyphal tip isolation. For molecular confirmation, a portion of the translation elongation factor 1-alpha (EF1α) was amplified by PCR using EF1/EF2 primers (O'Donnell et al., 1998). Additionally, the RNA polymerase subunit RPB2 was amplified as two contiguous fragments via primers and protocols described by O'Donnell et al., 2021. Amplicons were sequenced (GenBank Accession Nos. PP426617 - 426626, PQ058494 - 058513). Using Fusarium-ID and Fusarioid-ID databases, EF1α and RPB2 sequences were found to be more than 99% identical to published F. oxysporum type isolates and Fusarium sp. isolates in the F. oxysporum species complex (Crous et al., 2021; Wang et al., 2022). A specific F. oxysporum f. sp. fragariae (Fof) qPCR assay (Burkhardt et al., 2019) was used to determine if these isolates could be Fof race 1. A Fof race 1 isolate (MAFF305558), negative controls, and water controls were included. All ten isolates and the Fof race 1 control were positive (Ct < 30), while other Fusarium spp. used as negative controls and the water controls did not amplify. Two isolates (F.200 and F.202) and MAFF305558 (positive control) were included in a pathogenicity test on two strawberry cultivars, 'Monterey' (susceptible to race 1) and 'Fronteras' (resistant to race 1) (Dilla-Ermita et al., 2023). Each isolate was included in two independent trials. In each trial, 5 plants per cultivar were inoculated by dipping roots for 10 min in 5 × 106 conidia/mL of 0.1% water agar (WA) or in sterile 0.1% WA for the negative control plants. Each plant was then planted in a pot filled with peat. Pots were placed randomly in random positions in a growth chamber at 28/20°C and 12h photoperiod. After 8 - 10 weeks, the control plants and 'Fronteras' plants remained healthy, while the inoculated plants cv. 'Monterey' were severely wilted and/or dead. Fusarium oxysporum was re-isolated from all symptomatic plants. Recovered isolates were confirmed to be the same as the inoculated ones using the Fof-qPCR, including the same controls as above. This is the first report of Fof race 1 in the Iberian Peninsula. Given that the land was not previously used for strawberry, it is highly probable that the pathogen was introduced with the planting material originated from a Spanish nursery. In conclusion, it is imperative to implement more severe control measures in nurseries to avoid the spread of this race.

Postharvest fruit rot on red guava caused by Neopestalotiopsis saprophytica newly reported in Hainan Province China

Red Guava is widely grown in numerous parts of southern China and is a favorite fruit among Chinese consumers due to its imperative nutritional value, high medicine value and excellent economic value. Black spot is one of the serious diseases that causes fruits rot. In July 2023, 25–30% of postharvest red guava fruits rot exhibiting disease symptoms were observed in surrounding markets of Haikou city, Hainan province, China. The purpose of this study was to isolate and identify the pathogenic fungi of red guava black spot. A total of 26 pathogenic fungal strains were isolated from the rotten red guava fruits with typical characteristics. One representative isolate, FSL2, was selected for subsequent experiments. Combining morphological analysis with phylogenetic analysis (internal transcribed spacer regions 4 and 5, translation elongation factor1, and β-tubulin genes), the pathogens should be identified as Neopestalotiopsis saprophytica. Pathogenic tests indicated that the symptoms of red guava fruit decay caused by N. saprophytica isolated from the sample were almost the same. The fungal species has been previously reported in China on Persimmon related with fruits rot. Thus, this study concluded that the pathogen of red guava black spot may be N. saprophytica. To best of our knowledge, this is the first report of red guava black spot caused by N. saprophytica.

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

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

First report of Fusarium luffae causing soybean wilt in the USA.

During the 2023 soybean growing season in South Dakota, we scouted a farmer's field and observed soybean (Glycine max (L.) Merr.) plants with wilting symptoms and blighted leaves. Symptomatic stems and leaves were collected from the field to identify associated pathogens. 0.5 cm2 size leaf and stem segments of the sample were surface sterilized by rinsing with 10% bleach for 5 minutes then dipping in 70% ethanol for one minute, and later placing in deionized sterile water for one minute. The sterilized segments were placed on wet filter paper and incubated under fluorescent light for three days. Fungal growth was observed, and the growing mycelia were transferred to potato dextrose agar plates amended with 50 µg/ml Ampicillin (PDAa). Pure culture of the isolate was obtained using single sporing and transferring on new PDAa plates. A dense aerial mycelial growth showing waxy yellow color with a pale orange tinge on the rear side covered the full plate after seven days of incubation at room temperature under fluorescent lights (Figure S1a and b). Developing macroconidia were falcate, curved, smooth to slightly rough, and hyaline with three-five septa (Figure S1c). For molecular identification, DNA of the recovered isolate was extracted and subjected to multiloci PCR (O'Donnell et al., 2010) to amplify and Sanger sequence the internal transcribed spacers region (ITS) (GenBank accession number PP393518), calmodulin (CAM-PP401978), RNA polymerase II second largest subunit (RPB2-PP401980), and translation elongation factor 1-α gene (TEF1-PP401979). The South Dakota isolate (SLSDF2) was identified as Fusairum luffae on NCBI and Fusarioid polyphasic identification databases with 99.40% similarity to Fusarium luffae strain NRRL31167. A phylogeny was inferred based on concatenated TEF1, RPB2, and CAM sequences to show species relatedness (Figure S3). The characterized isolate SDSLF2 was evaluated for soybean pathogenicity using spray inoculations on detached leaves and V2 stage soybean plants (Figure S2a and b). The conidial suspension was prepared by growing the pathogen on mung bean agar for seven days. 2 ml of conidial suspensions (2.6 × 104 conidia/ml) and mock control (sterilized water with 0.1% Tween-20) was sprayed on the detached leaves and whole plants. The experiment was repeated three times with four replicates in each. In the detached leaf assay, leaves were completely blighted (Figure S2a) within 96 hours. In whole plant assays, after two days of incubation, leaf blighting was visible and progressed with time. Four days post-inoculation, the infected plants showed extensive leaf symptoms, and ultimately defoliation occurred (Figure S2b). No symptoms were observed in mock controls of either of the experiments. The pathogen was reisolated from the infected tissues and its identity was confirmed as F. luffae by CAM sequencing fulfilling Koch's postulates. F. luffae has been reported to associated with soybeans in China (Zhao et al., 2022), however, to our knowledge, this is the first report of F. luffae pathogenic on soybeans in the USA, stressing the need to identify resistance sources to avoid any potential disease epidemic.

Unmasking of molecular players: proteomic profiling of vitreous humor in pathologic myopia

BackgroundThis study aimed to identify the differentially expressed proteins in the vitreous humor (VH) of eyes with and without pathologic myopia (PM), providing insights into the molecular pathogenesis.MethodsA cross-sectional, observational study was conducted. VH samples were collected from patients undergoing vitrectomy for idiopathic epiretinal membrane (ERM), macular hole (MH), or myopic retinoschisis (MRS). Label-free quantitative proteomic analysis identified differential protein expression, with validation using ELISA.ResultsThe proteomic profiling revealed significantly higher expressions of tubulin alpha 1a (TUBA1A) and eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) in PM groups (MH-PM, MRS-PM) compared to controls (MH, ERM). Conversely, xylosyltransferase 1 (XYLT1), versican core protein (VCAN), and testican-2 (SPOCK2) expressions were lower in PM. ELISA validation confirmed these findings.ConclusionsOur study provides novel insights into the molecular mechanisms of PM. The differentially expressed proteins EEF1A1, TUBA1A, XYLT1, VCAN, and SPOCK2 may play crucial roles in chorioretinal cell apoptosis, scleral extracellular matrix (ECM) synthesis, and scleral remodeling in PM. These proteins represent potential new targets for therapeutic intervention in PM, highlighting the importance of further investigations to elucidate their functions and underlying mechanisms in disease pathogenesis.

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

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

Diversity and pathogenicity of Fusarium spp. isolated from cultivated sorghum stems and roots in eastern Australia

AbstractStalk and root rots of cultivated sorghum (Sorghum bicolor) are caused by several Fusarium species worldwide. This study evaluated Fusarium diversity, pathogenicity and population structure amongst 212 isolates obtained from 169 sorghum plants from commercial field crops in eastern Australia. Sequences of translation elongation factor‐1α (tef‐1α), RNA polymerase II largest subunit (rpb1), RNA polymerase II second largest subunit (rpb2) and calmodulin (cmdA) were used to construct multilocus phylogenies that enabled the identification of 16 Fusarium species in Fusarium chlamydosporum species complex (FCSC), Fusarium fujikuroi species complex (FFSC), Fusarium incarnatum‐equiseti species complex and Fusarium oxysporum species complex (FOSC). The majority of isolates (n = 171) belonged to FFSC. The pathogenicity of 17 selected isolates was determined by artificial inoculation of sorghum seedlings and completing Koch's postulates. Isolates of species in FFSC were significantly (p &lt; 0.05) more aggressive as root pathogens in sorghum seedlings than isolates of other species tested and widely distributed across all sampling sites. Amongst the 35 isolates of FOSC, 26 belonged to Fusarium cili, which is only known as an endophyte from healthy roots of Rosa roxburghii in China. Fusarium sporodochiale (in FCSC) and Fusarium contaminatum (in FOSC) are reported as sorghum seedling root rot pathogens for the first time.

Fusarium andiyazi, a Pathogenic Species Associated with Rice Bakanae Disease in Malaysia.

Rice is the main staple food crops for the Malaysian population. Rice is also susceptible to bakanae diseases caused by some Fusarium species and reducing yield, and quality of rice also profit. In this study, several rice fields were surveyed to collect Fusarium isolates associated with bakanae disease. The morphological features of Fusarium andiyazi isolates found on infected rice plants were identified in this investigation. For biological species identification, MAT-1 (Mating type idiomorphs) bearing isolates were crossed with MAT-2 isolates. Crossing was succeeded between cross of two different mating type bearing field isolates. Consequently, there is a possibility of exchange of genetic material within the F. andiyazi population in Malaysia. The identity of the isolates was further determined up to the species level by comparing DNA sequences and phylogenetic analysis of two genes. The phylogenetic analyses of the joined dataset of translation elongation factor 1-alpha (TEF1-α) and RNA polymerase subunit II (RPB2) revealed that all the isolates were F. andiyazi. In pathogenicity tests, F. andiyazi were found to be pathogenic on the susceptible rice cultivars MR211 and MR220. Inoculated rice seedling produced typical bakanae symptom like elongation, thin and yellow leaves. F. andiyazi was further confirmed as pathogenic species by Ultra High-Performance Liquid Chromatography (UPLC) detection of Gibberellic acid (GA3) and Fusaric acid. In this study, F. andiyazi strains have been identified as the responsible pathogen for causing rice bakanae disease in Malaysia and it is the first report of F. andiyazi, as a pathogenic species on rice in Malaysia.

Diversity and Pathogenicity of Six Diaporthe Species from Juglans regia in China.

Walnut (Juglans regia L.) is cultivated extensively in China for its substantial economic potential as a woody oil species. However, many diseases caused by Diaporthe greatly affect the health of Juglans regia trees. The present study revealed the presence of Diaporthe species from Juglans regia. A total of six species of Diaporthe were isolated from twigs of Juglans regia in three provinces in China, including two known species (Diaporthe gammata and D. tibetensis) and four novel species (D. chaotianensis, D. olivacea, D. shangluoensis and D. shangrilaensis). Phylogenetic relationships of the new species were determined by multilocus phylogenetic analyses based on partial sequences of the internal transcribed spacer (ITS) region, calmodulin (cal) gene, histone H3 (his3) gene, translation elongation factor 1-α (tef1-α) gene and β-tubulin (tub2) gene. Pathogenicity tests indicated that all Diaporthe species obtained in this study were confirmed as pathogens of Juglans regia. This study deepens the understanding of species associated with several disease symptoms in Juglans regia and provides useful information for effective disease control.

Neopestalotiopsis rosae, a novel pathogen causing leaf blight and crown rot of strawberries in India

Neopestalotiopsis have been reported to affect sustainable strawberry production across the world. Strawberry leaves with leaf blight symptoms were collected in January 2023 from the nurseries and fields of Department of Horticulture, Gandhi Krishi Vigyan Kendra, GKVK, Bangalore, India. Black conidiomata with slimy black conidial mass was produced upon incubating at 28 ± 2 °C. Conidia were fusoid to ellipsoidal, and comprised five cells. The middle three cells were versicolored. Whereas, the apical and basal cells harboured three to four and one appendages, respectively. Neopestalotiopsis rosae was confirmed based on sequencing of internal transcribed spacer (ITS) region of rDNA, β-tubulin (TUB2) and translation elongation factor 1-alpha (EF1-α) gene regions and phylogenetic analysis. Pathogenicity tests confirmed that Neopestalotiopsis rosae as the inciting agent of leaf blight and crown rot disease in strawberry posing a threat to strawberry production in India.

Isolation and identification of Cladosporium tenuissimum causing leaf blight on garden pea (Pisum sativum L.) in Telangana from southern India

Garden pea (Pisum sativum L.), is an important legume grown worldwide as a source of proteins and carbohydrates. Since pea crop is cultivated globally in cool temperate climatic conditions, increasing temperatures accompanied by high humidity favors fungal infections. A fungal infection causing leaf blight symptoms was frequently observed on the leaves of pea plants grown in greenhouse and field conditions. The blight symptoms showed a water-soaked and wilting appearance with curly leaf edges accompanied by greyish mycelial growth and chlorotic lesions. The disease incidence in field conditions ranged between 5 and 10 %, whereas in the controlled greenhouse conditions, it ranged from 40 to 45%. Two isolates of the fungus, Gp03 and Gp04 were isolated from Arkel pea leaves and purified from a single conidium each. According to Koch's postulates, the pathogenicity tests with these isolates using detached leaf assay and whole plant leaf assays confirmed the pathogen. Morphological and molecular analysis of nucleotide sequences of the D1-D2 region, translation elongation factor-1α (TEF) and actin (ACT) genes confirmed these isolates as members of the genus, Cladosporium. The phylogenetic relatedness of these isolates with other members of the Cladosporium genus revealed them as fungal strains of Cladosporium tenuissimum. Further, these isolates were also confirmed as C. tenuissimum by Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh in India, and provided the accession nos. MTCC 13581 and MTCC 13582 to the C. tenuissimum strains, Gp03 and Gp04 respectively. This is the first report of Cladosporium tenuissimum infecting garden pea in Telangana, India.

Altering translation allows E. coli to overcome chemically stabilized G-quadruplexes.

G-quadruplex (G4) structures can form in guanine-rich DNA or RNA and have been found to modulate cellular processes including replication, transcription, and translation. Many studies on the cellular roles of G4s have focused on eukaryotic systems, with far fewer probing bacterial G4s. Using a chemical-genetic approach, we identified genes in Escherichia coli that are important for growth in G4-stabilizing conditions. Reducing levels of elongation factor Tu or slowing translation elongation with chloramphenicol suppress the effects of G4 stabilization. In contrast, reducing expression of certain translation termination or ribosome recycling proteins is detrimental to growth in G4-stabilizing conditions. Proteomic and transcriptomic analyses demonstrate that ribosome assembly factors and other proteins involved in translation are less abundant in G4-stabilizing conditions. Our integrated systems approach allowed us to propose a model for how RNA G4s can present barriers to E. coli growth and that reducing the rate of translation can compensate for G4-related stress.