EF-1α Gene Research Articles

First Report of Root Rot Caused by Fusarium equiseti on Syrian rue (Peganum harmala) in China

Syrian rue (Peganum harmala L) belongs to the family Zygophyllaceae and is mainly distributed in arid and semi-arid areas of Xinjiang, Gansu, Ningxia, Qinghai, and Inner Mongolia in China. It serves as a pioneer species in soil and water conservation, as well as in reclamation of wastelands, playing a crucial role in soil preservation and stabilization against sand encroachment. In 2023, a disease resembling root rot and wilt affected the quality and yield of Syrian rue in Xilingol (122.67°N, 42.78°E), Inner Mongolia, China. Symptoms ranged from yellowing to wilting, accompanied by darkening of the xylem in the roots. This disease was observed in approximately 50% of Syrian rue plants in desertification-prone grassland. A total number of 45 symptomatic root samples were collected from Syrian rue plants to isolate the pahogen. Small pieces of diseased tissue were surface sterilized with 75% alcohol for five seconds, rinsed twice with sterilized water, dried, and then placed on water agar and incubated for 72 h at 25℃ in darkness. A total of 45 isolates were obtained, showing identical morphology and sequences of the internal transcribed spacer region (ITS), translation elongation factor 1-alpha (EF1-α) and RNA polymerase II second largest subunit (RPB2) genes. Therefore, the isolate LTP-5 was used as the representative for further study. On potato dextrose agar (PDA), fungal colonies of LTP-5 exhibited yellow to light brown colore with white aerial mycelia and irregular growth after 7 days of incubation at 25℃. Conidia and chlamydospre development were observed in carboxymethylcellulose sodium medium. Chlamydospores were abundant, terminal or intergrown between hyphae, rough brown walls of 5.9 to 19.82 μm in diameter (n = 75) were observed in the culture. Macroconidia had 5 to 7 septa, thick in the middle and thin at both ends, measured 28.17 to 62.81 μm in length and 1.50 to 4.19 μm in width (n = 128). Microconidia were absent. The morphological characteristics were consistent with the descriptions of Fusarium equiseti (Nelson et al., 1983). The pathogen was further confirmed to be F. equiseti by sequence analysis of the ITS, EF1-α and RPB2 genes amplified using polymerase chain reaction (PCR) with the primers ITS4/ITS5 (White et al., 1990), EF-1/EF-2 (O'Donnell et al., 1998), and RPB2-5F2 /fRPB2-7cR (Zhen et al., 2017). The sequences showed 100% similarity to the F. equiseti strain in the NCBI GenBank with accession numbers MH054914.1, KJ396323.1, and KT213286.1 for ITS, EF1-α, and RPB2, respectively. The sequences of PCR products were submitted to GenBank with accession numbers PP814863.1 (ITS), PP831628.1 (EF1-α) and PP826937.1 (RPB2). Pathogenicity tests were conducted through a pot assay. Five Syrian rue seedlings were inoculated by immersing plant roots in a suspension of 1×107 spores/mL for 30 min and transplanting to pots containing autoclaved soil. Control plants were immersed in sterile water. The inoculated plants showed yellow and root rot but no symptoms were observed on control plants. The same fungus was successfully re-isolated and identified based on the morphological characterization and sequence analyses from the infected root tissue but not from non-inoculated plants. In addition, F. equiseti has been reported to cause disease on Sugar beet (Khan et al., 2021), Watermelon (Rahman et al., 2021), potato (Cui et al., 2021), and other plants. This is the first report confirming F. equiseti as causal agent of Syrian rue wilt and root rot in China.

A new species of Diacheopsis (Myxomycetes) and a new habitat for myxomycetes

ABSTRACT We describe a new species, Diacheopsis resinae (Myxomycetes), collected from a microhabitat new for myxomycetes: stem wounds of coniferous trees (Norway spruce) where the resin is overgrown with a community of resinicolous fungi. The 80 known collections come from the Vosges (France), the Black Forest (Germany), Swabian Alp (Germany), and several localities in Denmark and Norway. Observations, but as well as metabarcoding of substrate samples with fungal (ITS [internal transcribed spacer]), bacterial (16S rDNA), and myxomycete (18S nuc rDNA) primers from eight trunks, revealed the new myxomycete to co-occur with resin-degrading ascomycetes (Infundichalara microchona, Lophium arboricola, Zythia resinae). The gram-negative bacterial genera Endobacter and Sphingomonas were found to be abundant in the substrate and may be a food source for the myxomycete. Fruit bodies were found mostly during the more humid winter season, with a peak in January/February. Partial sequences of two independent molecular markers (18S nuc rDNA, EF1α [elongation factor 1-alpha] gene) were obtained for 41 accessions, which form a monophyletic cluster in a two-gene phylogeny of Stemonititidales but do not group with other species of Diacheopsis, thus rendering this genus paraphyletic. The new species, although exclusively developing sessile sporocarps and morphologically undoubtedly falling into the genus Diacheopsis, is most closely related to species of Lamproderma, especially L. album, L. zonatum, and L. zonatopulchellum. Within D. resinae, three groups can be differentiated, which show nearly complete reproductive isolation, as judged from a recombination analysis of the two unlinked markers and the allelic combinations of the EF1α gene.

Identification, characterization, and sensitivity to phytochemicals of a novel Curvularia species associated with leaf spot disease on Curcuma kwangsiensis

A leaf spot disease affecting Curcuma kwangsiensis (Zingiberaceae) has been observed in Qinzhou City, Guangxi Province. Infected leaves exhibit yellow-brown spots that progressively expand and eventually lead to leaf death. Curvularia isolates were obtained from the diseased leaves with tissue isolation and single spore purification methods. To accurately identify these isolates, we analyzed their morphological characteristics and phylogenetic relationships using combinations of ITS, GAPDH, and EF-1α gene sequences. Phylogenetic analysis showed that the investigated strains formed a distinct clade separate from other recognized Curvularia species. Furthermore, the strains exhibited differences in conidiophore size and conidia shape/size. Based on phylogenetic studies, morphology, and pathogenicity tests, the pathogen was identified as a new species named Curvularia qinzhouensis. Optimal conditions for mycelial growth were observed at 30 °C and pH 8. The sensitivity of the pathogen to various phytochemicals was also examined. Honokiol, thymol, and citral demonstrated effective antifungal effects, with EC50 values of 6.72 ± 1.75, 25.74 ± 4.30, and 54.24 ± 4.69 µg/ml, respectively. The present investigation provides the first report of leaf spot disease on C. kwangsiensis caused by C. qinzhouensis, and valuable insights for the prevention and control of this disease.

The terrestrial flatworm Microplana scharffi (Geoplanidae, Microplaninae): mitochondrial genome, phylogenetic proximity to the Bipaliinae and genes related to regeneration

A genome skimming approach of sequencing was undertaken on a subfamily of terrestrial flatworms that had been neglected in genomic studies until now, namely the Microplaninae as represented here by Microplana scharffi. A single run of short-read sequencing enabled retrieval of the complete mitogenome, the two paralogous versions of the 18S gene, the elongation factor gene EF1α, plus two genes involved in the regeneration process, namely those coding for ß-CATENIN-1 and adenomatous polyposis coli (APC). The 15,297 bp mitogenome lacks a functional tRNA-Ala and has a mandatory alternative TTG start codon in its cox1 gene. The multiprotein phylogeny, inferred from mitogenome proteins, positions M. scharffi as sister-group to the Bipaliinae with maximum support, although the organisation of the mitogenomes shows features previously never observed among Bipaliinae, such as the conserved 32 bp overlap between ND4 and ND4L. Similarly to what has been observed in recent publications on other species of Geoplanidae, the two types of 18S genes display strongly different coverages and are only 90.57% identical. Additionally, alien DNA was identified in the pool of contigs in the form of the complete mitochondrial genome of Lumbricus rubellus, confirming previous observations on the feeding habits of M. scharffi.

A new species of Craterium (Myxomycetes, Physarales, Physaraceae) with a mottled peridium

A new species of Craterium, described herein as С. guttatum, was recovered in Russia, during intensive field work in the Kaluzhskiye Zaseki Nature Reserve (Kaluga Oblast’) and in the Sudogodskiy District of the Vladimir Oblast’. Morphological details of sporocarps and spores were examined by light and scanning electron microscopy. The new species is characterized by a unique combination of morphological characters, such as a beaker-shaped sporotheca with a mottled peridium and a prominent line of dehiscence separating the upper part of the sporotheca in the form of a convex white lid, as well as spores densely ornamented with small warts grouped in clusters. The partial sequences of the 18S rRNA, EF1α and mtSSU genes of C. guttatum differ significantly from all Craterium species sequences available to date.

Identification of hub genes and potential networks by centrality network analysis of PCR amplified Fusarium oxysporum f. sp. lycopersici EF1α gene

BackgroundFusarium wilt is a devastating soil-borne fungal disease of tomato across the world. Conventional method of disease prevention including usage of common pesticides and methods like soil solarisation are usually ineffective in the treatment of this disease. Therefore, there is an urgent need to identify virulence related genes in the pathogen which can be targeted for fungicide development.ResultsPathogenicity testing and phylogenetic classification of the pathogen used in this study confirmed it as Fusarium oxysporum f. sp. lycopersici (Fol) strain. A recent discovery indicates that EF1α, a protein with conserved structural similarity across several fungal genera, has a role in the pathogenicity of Magnaporthe oryzae, the rice blast fungus. Therefore, in this study we have done structural and functional classification of EF1α to understand its role in pathogenicity of Fol. The protein model of Fol EF1α was created using the template crystal structure of the yeast elongation factor complex EEF1A:EEF1BA which showed maximum similarity with the target protein. Using the STRING online database, the interactive information among the hub genes of EF1α was identified and the protein–protein interaction network was recognized using the Cytoscape software. On combining the results of functional analysis, MCODE, CytoNCA and CytoHubba 4 hub genes including Fol EF1α were selected for further investigation. The three interactors of Fol EF1α showed maximum similarity with homologous proteins found in Neurospora crassa complexed with the known fungicide, cycloheximide. Through the sequence similarity and PDB database analysis, homologs of Fol EF1α were found: EEF1A:EEF1BA in complex with GDPNP in yeast and EF1α in complex with GDP in Sulfolobus solfataricus. The STITCH database analysis suggested that EF1α and its other interacting partners interact with guanosine diphosphate (GDPNP) and guanosine triphosphate (GTP).ConclusionsOur study offers a framework for recognition of several hub genes network in Fusarium wilt that can be used as novel targets for fungicide development. The involvement of EF1α in nucleocytoplasmic transport pathway suggests that it plays role in GTP binding and thus apart from its use as a biomarker, it may be further exploited as an effective target for fungicide development. Since, the three other proteins that were found to be tightly associated Fol EF1α have shown maximum similarity with homologous proteins of Neurospora crassa that form complex with fungicide- Cycloheximide. Therefore, we suggest that cycloheximide can also be used against Fusarium wilt disease in tomato. The active site cavity of Fol EF1α can also be determined for computational screening of fungicides using the homologous proteins observed in yeast and Sulfolobus solfataricus. On this basis, we also suggest that the other closely associated genes that have been identified through STITCH analysis, they can also be targeted for fungicide development.

First Report of Diaporthe hongkongensis Causing Leaf Blight on Macadamia in China.

Macadamia (Macadamia ternifolia Maiden and Betche) belongs to the Proteaceae family (Li et al. 2022). In the hilly areas of Guangxi (southern China), macadamia trees are an important source of revenue. The planting area in Guangxi has increased in recent years, exceeding 53,333 hectares by the end of 2022, but this increase is also associated with emergency of, macadamia diseases. Leaf blight symptoms were observed in 37/241 macadamia trees (15% incidence) in a plantation in Nanning, Guangxi province in China, during June, 2022. Disease severity on infected trees ranged from 5% to 60%. The disease developed from the tips or margins of leaves, causing the leaves to turn brown, and later gradually withered (Fig. 1 A). Ten leaves with lesions were collected from five macadamia trees (two leaves per tree. Thereafter, small segments (3 to 4 mm²) excised from the margins of ten lesions were surface sterilized in 75% ethanol for 30 s and 1% hypochlorite for 90 s and Page 1 of 6 2 rinsed in sterile water, before plating onto potato dextrose agar (PDA) medium. Plates were incubated under lighting during the daytime, and darkness at night-time for 5 days at 25℃. Twenty-two purified colonies were generated by subculturing hyphal tips, of which eight exhibited similar morphology and were further characterized. The colonies on PDA were gray with a white outer ring and flat lawn on the surface (Fig. 1 B). The pycnidia were superficial to semi-immersed on PDA, solitary to aggregated, globose to sub-globose, brown to black and oozed yellow mucilaginous masses (Fig.1 C). The α-conidia were unicellular, hyaline elliptical or fusiform, and measuring 4-8 × 1.9-4 μm (n=30) ， whereas the β-conidia were hyaline, long, straight or curved, measuring 20-23 × 0.9-2 μm (n=30) (Fig. 1 D-E). The morphological features were similar to Diaporthe hongkongensis (Dissanayake et al. 2015). The eight morphologically similar isolates were identified as D. hongkongensis using the internal transcribed spacer (ITS) region, but only one isolate, JG11, was selected for further molecular identification. Five target genes, including the ITS region, translation elongation factor 1 alpha (EF1-α), beta-tubulin genes (TUB2), calmodulin (CAL), and histone H3 (HIS) were amplified and sequenced using primers ITS1/ITS4, EF1-728F/EF1-986R, Bt2a/Bt2b, CAL-228F/CAL-737R, and CYLH3F/H3-1b, respectively (Carbone and Kohn 1999). The sequences were deposited in GenBank under accession numbers OQ932790 (ITS) and OR147955-58 for EF1-α, TUB, CAL and HIS genes, respectively. BLAST search of GenBank showed that ITS, EF1-α, TUB, CAL, and HIS sequences of JG11 were similar to Page 2 of 6 3 those of D. hongkongensis NR111848 (99.22% identity), KY433566 (99.72%), MW208603 (99.42%), MW221740 (99.80%), and MW221661 (99.79%), respectively. Phylogenetic analysis of concatenated sequences was performed with IQ-TREE software. JG11 was grouped in the same clade as other Diaporthe hongkongensis isolates (Fig. 2). Pathogenicity experiments were carried out on healthy macadamia trees in a greenhouse. Three macadamia trees were used as negative controls where five uninjured leaves per tree were sprayed with sterile distilled water. Uninjured five leaves per tree of three other macadamia trees were sprayed with conidia suspension of the isolate JG11 at a concentration of 1×106. Each treatment was repeated 3 times independently, with 5 leaves per tree (Liu et al. 2023; Havill et al. 2023; Zhang et al. 2022). Plastic bags were placed over all inoculated leaves. The average daily temperature and relative humidity in the greenhouse were 32°C and 65%, respectively. Two days later, browning appeared on the leaves inoculated with the spore suspension and expanded outward. After 5 days, all macadamia leaves inoculated with the fungal spores began to wither, while controls remained asymptomatic (Fig. 1 H-I). D. hongkongensis was consistently re-isolated and purified from inoculated leaves and the identity was confirmed by morphological identification and molecular analysis, completed Koch's postulates. D. hongkongensis has been reported on peach (Zhang et al. 2021), grapevine trunk (Dissanayake et al. 2015) and Cunninghamia lanceolata (Liao et al. 2022). To our knowledge, this is the first report of D. hongkongensis causing leaf blight on macadamia in China. These findings provide a foundation for future research on the epidemiology and control of this newly emerging disease of macadamia.

Development of a Multiplex Real-Time PCR to Disambiguate Culicoides sonorensis within Culicoides variipennis Complex, the Proven Vector of Bluetongue and Epizootic Hemorrhagic Disease Viruses in North America.

Species delimitation of Culicoides complex species can be challenging. Among species within the Culicoides variipennis complex, C. sonorensis is considered the primary vector of bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in North America. Morphological identification of C. sonorensis within the C. variipennis complex is laborious, time-consuming, and requires entomology expertise. Therefore, in this study we developed and validated a multiplex real-time PCR for rapid detection and differentiation of C. sonorensis from the two other main cryptic species (C. variipennis and C. occidentalis) within the C. variipennis complex. The assay targets the EF1α gene and has a built-in internal control targeting 18 S. The specificity and the sensitivity of the multiplex real-time PCR were evaluated using morphologically identified reference and field-collected specimens. The multiplex PCR was 100% specific when nucleic acid extracted from C. variipennis, sonorensis, and occidentalis specimens was tested. When nucleic acid extracted from pools of midges was tested, the multiplex PCR was able to detect all three Culicoides species with comparable sensitivity. The multiplex assay, however, failed to detect eight morphologically identified C. sonorensis specimens collected from Alberta in 2014. The EF1α gene sequences of these specimens formed a distinct phylogenetic cluster, amongst those from C. variipennis, sonorensis, and occidentalis, suggesting that they belong to a different species. We hypothesize that those specimens might be C. albertensis, the only other species remaining in the C. variipennis complex with known geographical distribution in North America. We believe that this highly sensitive and specific multiplex real-time PCR assay could be an effective tool for rapid detection and differentiation of C. sonorensis, the known vector of BTV and EHDV, in trap collections in future vector surveillance programs.

First Report of Basal Stem Rot of Achnatherum inebrians Caused by Fusarium pseudograminearum in China.

Drunken horse grass (Achnatherum inebrians) is a perennial bunchgrass that is widely distributed in arid and semi-arid grasslands in northwest China (Zhang et al., 2021). In July 2023, Basal stem rot was found in artificially grown drunken horse grass plots in Yuzhong County (35.85° N, 104.12° E), Gansu Province, China, with an average incidence of 5.2%. Diseased plants showed crown and basal stem rot with chocolate brown discoloration at the base of the stem and slight constriction of some basal stems. Five field's foci were surveyed and at least 6 basal stems per focus were collected. Infected basal stems were surface-sterilized (75% ethanol for 30 s and 1% NaClO for 90 s), rinsed three times with sterilized water, placed on potato dextrose agar (PDA), and incubated at 22°C in the dark for 3 days. Isolates were purified by single spore cultures (Leslie and Summerell, 2006). The average mycelial growth rate was 4.8 to 7.5 mm/day at 25°C on PDA, and the colonies produced aerial mycelium varying from rose to yellow white, and rose to burgundy pigment diffused into the agar. Macroconidia of the isolates were produced on carnation leaf agar (CLA) incubated under black light and observed to be abundant, but no microconidia were found. Macroconidia were relatively slender, curved to almost straight, commonly 3-6 septate, averaging 30.1 × 3.8 μm (n=50). The morphological characteristics of this fungus fully fit the description of F. pseudograminearum (Aoki and O'Donnell, 1999). To obtain the phylogenetic support, DNA of three representative isolates YZ-Y-1, YZ-Y-2 and YZ-Y-3 was extracted by using an HP Fungal DNA Kit (D3195), and a portion of the RNA polymerase II second largest subunit (RPB2) gene and elongation factor 1 alpha (EF1-α) gene were amplified using primers RPB2-5f2 and RPB2-7cr (O'Donnell et al. 2010) and EF1 and EF2 (O'Donnell et al. 1998), respectively. Results of sequences were deposited in GenBank (accession nos. PP179044 to PP179049). A nucleotide BLAST search revealed RPB2 and EF1-α sequences to be 99.8 and 100% similar to the corresponding sequences of the ex-type strain NRRL 28062 F. pseudograminearum accessions numbers MW233433 and MW233090, respectively. For pathogenicity tests, 15 μl of conidia suspension (1×106 conidia/ml) was inoculated into the stem bases of 10 healthy drunken horse grass seedlings (around 3 weeks old) using a sterile syringe, then wrapped with moistened sterile gauze, while the other 10 drunken horse grass seedlings were injected with sterile water as a control. All seedlings were placed in a greenhouse with a plastic cover at 15-22°C and 90-100% relative humidity. All inoculated drunken horse grass seedlings showed symptoms similar to those of natural infection with stem basal rot, whereas uninoculated drunken horse grass seedlings remained healthy after 15 days. Fungi re-isolated from the basal stems of inoculated plants were confirmed phenotypically and molecularly as F. pseudograminearum. To our knowledge, this is the first report of F. pseudograminearum causing crown rot of drunken horse grass in China. The disease has become a potential threat to the growth of drunken horse grass in China.

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.

Novel 3D human trophoblast culture to explore T. cruzi infection in the placenta.

Human trophoblastic cell lines, such as BeWo, are commonly used in 2D models to study placental Trypanosoma cruzi infections. However, these models do not accurately represent natural infections. Three-dimensional (3D) microtissue cultures offer a more physiologically relevant in vitro model, mimicking tissue microarchitecture and providing an environment closer to natural infections. These 3D cultures exhibit functions such as cell proliferation, differentiation, morphogenesis, and gene expression that resemble in vivo conditions. We developed a 3D culture model using the human trophoblastic cell line BeWo and nonadherent agarose molds from the MicroTissues® 3D Petri Dish® system. Both small (12-256) and large (12-81) models were tested with varying initial cell numbers. We measured the diameter of the 3D cultures and evaluated cell viability using Trypan Blue dye. Trophoblast functionality was assessed by measuring β-hCG production via ELISA. Cell fusion was evaluated using confocal microscopy, with Phalloidin or ZO-1 marking cell edges and DAPI staining nuclei. T. cruzi infection was assessed by microscopy and quantitative PCR, targeting the EF1-α gene for T. cruzi and GAPDH for BeWo cells, using three parasite strains: VD (isolated from a congenital Chagas disease infant and classified as Tc VI), and K98 and Pan4 (unrelated to congenital infection and classified as Tc I). Seeding 1000 BeWo cells per microwell in the large model resulted in comparable cellular viability to 2D cultures, with a theoretical diameter of 408.68 ± 12.65 μm observed at 5 days. Functionality, assessed through β-hCG production, exceeded levels in 2D cultures at both 3 and 5 days. T. cruzi infection was confirmed by qPCR and microscopy, showing parasite presence inside the cells for all three tested strains. The distribution and progression of the infection varied with each strain. This innovative 3D model offers a simple yet effective approach for generating viable and functional cultures susceptible to T. cruzi infection, presenting significant potential for studying the placental microenvironment.

Evaluation of reference genes and expression patterns of CONSTANS-LIKE genes in Tetrastigma hemsleyanum under different photoperiods.

CONSTANS-LIKE (COL ) genes are a key signalling molecule that regulates plant growth and development during the photoperiod. Our preliminary experiments showed that the photoperiod greatly influence the formation of Tetrastigma hemsleyanum root tubers. In this study, we examined the oscillation patterns and expression characteristics of COL genes in leaves of T. hemsleyanum under different photoperiod conditions. Six genes were selected as candidate reference genes for further analyses: (1) 18S ribosomal RNA (18S rRNA ); (2) α-tubulin (TUBA ); (3) 30S ribosomal RNA (30S rRNA ); (4) TATA binding protein (TBP ); (5) elongation factor 1α (EF-1α ); and (6) RNA polymerase II (RPII ). The geNorm, NormFinder, and BestKeeper software programs were used to evaluate expression stability. Two ThCOL genes were screened in the T. hemsleyanum transcriptome library, and their expression patterns under different photoperiod conditions were analysed using quantitative reverse transcription PCR. The genes EF-1α , TUBA , and 18S rRNA were used to analyse the expression profiles of CONSTANS genes (ThCOL4 and ThCOL5 ) under different photoperiods. The expression peaks of ThCOL4 and ThCOL5 appeared at different times, demonstrating that their oscillation patterns were influenced by the photoperiod. We speculate that these two ThCOL genes may be involved in different biological processes.

Reference genes for RT-qPCR analysis in Musa acuminata genotypes contrasting in resistance to Fusarium oxysporum f. sp. cubense subtropical race 4

Banana (Musa spp.) is the most widely consumed fruit globally. Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is a highly threatening disease to banana production. Resistance genes to Foc exist in wild Musa genotypes such as Musa acuminata subsp. burmannicoides var. Calcutta 4. Whilst real-time PCR (RT-qPCR) is appropriate for accurate analysis of gene expression in pathways involved in host defence responses, reference genes with stable expression under specific biotic stress conditions and host tissue types are necessary for normalization of sample variation. In this context, the stability in potential host reference genes ACT1, APT, EF1α, GAPDH, αTUB, RAN, UBIQ1, UBIQ2, βTUB1, βTUB3, L2 and ACTA1 was evaluated in total RNA samples from root tissues in Calcutta 4 (resistant) and Musa sp. cultivar Prata-anã (susceptible) extracted during interaction with Foc subtropical race 4 (STR4). Expression stability was calculated using the algorithms geNorm, NormFinder and BestKeeper. βTUB3 and L2 were identified as the most stable in Calcutta 4, with ACTA1 and GAPDH the most stable in Prata-anã. These reference genes for analysis of gene expression modulation in the Musa-Foc STR4 pathosystem are fundamental for advancing understanding of host defence responses to this important pathogen.

Occurrence of Fusarium Crown Rot Caused by Fusarium culmorum on Winter Wheat in Xinjiang Uygur Autonomous Region, China.

Wheat (Triticum aestivum L.) is the predominant grain crop and plays a pivotal role in grain production in Xinjiang Uygur Autonomous Region (XUAR), China. Its cultivated area constitutes approximately half of the total sown area of grain crops in XUAR, with 1.14 million hectares in 2021. Fusarium crown rot (FCR) of wheat, caused by Fusarium culmorum (W.G. Smith) Sacc., is one of the most devastating soil-borne diseases known to seriously reduce grain yield (Ma et al. 2024; Saad et al. 2023). In 2016, FCR of wheat, caused by F. culmorum, was firstly identified in Henan Province, China (Li et al. 2016). In June 2023, during the investigation of FCR of wheat in Aksu Prefecture, XUAR, FCR on winter wheat (cv. Xindong 20) was found (82.761349°E, 41.612202°N). The grain-filling period for winter wheat in early June coincided with a period of high temperatures and water demand in Aksu Prefecture. Approximately 8% of the Xindong 20 wheat plants exhibited symptoms of white heads and browning at the stem base, with the disease present in 82% of the wheat fields surveyed. To identify the pathogens, 20 samples of diseased stem basal tissue, each 0.5 cm in length, were collected and sterilized with 75% alcohol for 30s and 5% NaOCl solution for 2 min, followed by three rinses with sterile water. These samples were then plated onto potato dextrose agar (PDA) medium at 25°C for 5 days. A total of 17 isolates with consistent morphological characteristics were obtained using single-spore technique, with an isolation rate of 85%. The isolated strains exhibited rapid growth on PDA, producing fluffy, pale-yellow hyphae, and accumulating a pale-yellow to dark red pigment on the bottom of the medium. On carnation leaf agar (CLA), these strains formed orange colonies due to the aggregation of a large number of macroconidia. The macroconidia were short and thick, with three to four septa and rounded apical cell, averaging 31.94 to 40.96 × 5.62 to 6.71 μm (Magnification of ×400). Microconidia were not observed. These morphological characters were consistent with those of F. culmorum (Leslie and Summerell. 2006). Two isolates (D-9 and D-11) were selected for molecular identification. The EF-1α gene fragment was amplified using primers EF1/EF2 (5'-ATGGGTAAGGARGACAAGAC-3'/5'-GGARGTACCAGTSATCATG-3') as previously described by O'Donnell et al. (1998). The two 665 bp PCR products were sequenced and submitted to GenBank (GenBank Accession No: PP763247 and PP763248) with 99. 7% identity to the published F. culmorum sequences (e.g., OP985478, OP985477, MG195126, KX702638). The molecular identification was further confirmed by F. culmorum species-specific PCR primers FcOIF/FcOIR (Nicholson et al. 1998). The expected PCR products of 553 bp were produced only in F. culmorum. Strains D-9 and D-11 were used to conduct the pathogenicity experiment on 7-day-old winter wheat (cv. Xindong 20) using drip in the lower stem inoculation method with a 10-μl of 106 macroconidia ml-1 suspension, and the control 7-day-old winter wheat were treated with sterile water (Xu et al. 2017). The experiments were replicated five times in a greenhouse at temperatures ranging from 20℃ to 25℃. After 4 weeks, all inoculated wheat seedlings showed stem base browning or even death. No symptoms were observed on the control plants. The fungus was reisolated from all inoculated wheat plants by the method described above and identified by morphological and PCR amplification using F. culmorum species-specific primers FcOIF/FcOIR. No F. culmorum was isolated from the control wheat plants, fulfilling Koch's postulates. To the best of our knowledge, this is the first report of F.culmorum causing FCR on winter wheat in XUAR, China. Considering wheat is the predominant grain crop and plays a pivotal role in grain production in China, necessary measures should be taken to prevent the spread of F. culmorum to other regions.

Characterization of Fusarium solani Associated with Tobacco (Nicotiana tabacum) Root Rot in Henan, China.

The aim of this study was to characterize the Fusarium solani species complex (FSSC) population obtained from tobacco roots with root rot symptoms by morphological characteristics, molecular tests, and assessment of pathogenicity. Cultures isolated from roots were white to cream with sparse mycelium on potato dextrose agar, with colony growth of 21.5 ± 0.5 to 29.5 ± 0.5 mm after 3 days. Sporodochia were cream on carnation leaf agar (CLA) and Spezieller Nährstoffarmer agar (SNA), and macroconidia formed in sporodochia were 3 to 6 septate and straight to slightly curved, with wide central cells, a slightly short blunt apical cell, and a straight to almost cylindrical basal cell with a distinct foot shape, ranging in size from 20.92 to 64.37 × 3.91 to 6.57 μm. Microconidia formed on CLA were reniform and fusiform, with 0 or 1 to occasionally 2 septa, that formed on long monophialidic conidiogenous cells, with a size range of 5.99 to 32.32 × 1.76 to 5.84 μm. Globose to oval chlamydospores were smooth- to rough-walled, 6.5 to 13.3 ± 0.37 μm in diameter, and terminal or intercalary and occurred singly, in pairs, or occasionally in short chains on SNA. Molecular tests consisted of sequencing and phylogenetic analysis of the translation elongation factor-1 alpha (EF-1α), RNA polymerase II largest subunit, and second largest subunit regions. All the obtained sequences revealed 98.14 to 100% identity to F. solani in both Fusarium ID and Fusarium MLST databases. Phylogenetic trees of the EF-1α gene and concatenated three-locus data showed that isolates from tobacco in Henan grouped in the proposed group 5, which is nested within FSSC clade 3 (FSSC 5). Twenty-seven of the 28 isolates caused root rot in artificially inoculated tobacco seedlings, with a disease severity index ranging from 15.00 ± 1.67 to 91.11 ± 2.22. Cross-pathogenicity tests showed that three representative isolates were virulent to six species of Solanaceae and two species of Poaceae, with disease severity indexes ranging from 6.12 ± 0.56 to 84.44 ± 0.00, indicating that these isolates have a wide host range. The results may inform the control of tobacco root rot through improved crop rotations.

First Report of Leaf Rot Caused by Apiospora paraphaeosperma on Lily in Wuhan, China.

Lily (Lilium spp.) is a valuable ornamental bulb flower plant in Liliaceae, and its bulbs have high edible and medicinal value. Compared with bulb propagation of other lilies, seed propagation and short growth period are the most significant characteristics of Lilium×formolongi. In 2023, leaf rot disease (LRD) was observed on approximately 70% of the Lilium×formolongi seedlings sown in an experimental greenhouse in Wuhan, Hubei province, China. Irregular brown water-soaked spots were discovered in the early stages of infected seedlings. Then, spots spread throughout the leaves and caused the leaves to brown, soften, and wilted. A pathogen associated with symptoms was isolated by incubating sterilized leaves on potato dextrose agar plates at 25 ℃ for 2-3 days. Then, a pure single colony was isolated through a single hyphal tip isolation method. The fungal colony was white with abundant aerial mycelium and produced a yellow pigment diffusible into the agar. Microscopically, isolated mycelia were reticulate and pale yellow, while conidia were dark brown, smooth, and spherical, 7.31 to 6.98 × 4.03 to 3.87μm (average 5.44×5.41μm; n=30); oval in lateral view, and had a light stripe in the middle. To identify the species of the fungus at the molecular level, ITS and EF-1α genes were amplified and sequenced using primers ITS1/ITS4 (M Gardes et al. 1993) and 758F/986R (Carbone and Kohn 1999). The BLAST results in GenBank showed that the ITS(OR523578) and EF-1α(PP066842) sequences of LRD shared 99.82% and 99.24% identity with the distinct Apiospora paraphaeosperma strains (GenBank accession MT040110, ON806628.1, respectively). Combined with the morphology of the colony and conidium, the fungus was identified as Ap. paraphaeosperma. In the pathogenicity test, six healthy leaves were inoculated with mycelium disc and then kept in an incubator (22 ℃, 90% humidity, 16h light /8h darkness). The inoculated leaves showed necrosis and wilt symptoms similar to those observed in the greenhouse, while the control leaves were asymptomatic. A re-isolation, morphology identification and DNA sequencing of the fungus confirmed its infection with Ap. paraphaeosperma in Lilium spp. At present, rot caused by Ap. paraphaeosperma has only been reported in Thailand and South Korea, both of which are found on bamboo stems (Hyde et al. 2016; Sun Lul Kwon et al. 2022). As far as we know, this is the first report of leaf rot of lily caused by Ap. paraphaeosperma in China. This report can help identify this disease and further develop effective control measures.

Characterization and Pathogenicity of Botryosphaeriaceae Species Associated with Gummosis, Dieback, Trunk and Branch Cankers of Almond Trees in Türkiye

Members of Botryosphaeriaceae family with 25 genera and several species are spread over a wide range of lands and climates worldwide. They cause gummosis, decline, dieback and blight on many woody plants. The purpose of present study was to diagnose the pathogens linked to the aforementioned symptoms on almond trees in seven orchards of Yozgat province (Türkiye) with a DSb type climate (Hot humid continental - Köppen Geiger system of climatic classification).These trees indicated and displayed dieback, gummosis trunk and branch canker symptoms. They were identified by cultural and morphological characteristics, and compared by phylogenetic sequencing of the ITS regions, EF-1α and β-tubulin genes with those of other species in GenBank (NCBI). Diplodia seriata, Lasiodiplodia theobromae, Neofusicoccum parvum and Botryosphaeria dothidea were identified in 72 isolates based on the colony and conidial characteristics. Successful pathogenicity tests were carried out on two-year-old almond seedlings of cv: Ferredual using Koch’s postulates. The results validated the identification According to available literature on the subject, identification of B. dothidea was done for the first time on almond trees in Türkiye. Accurate identification, prevalence and incidence of the pathogens are crucial for developing effective disease management strategies to arrest disease outbreaks in Türkiye.

Occurrence of Stem Rot on Acacia mangium Caused by Amauroderma subresinosum.

Acacia mangium has the characteristics of developed root system, nitrogen fixation and soil improvement, fast growth and high yield, and improvement of soil fertility. It is often used as a windbreak tree species in rubber plantations, a highway shade tree, for coastal and mountain restoration in Hainan . In October 2021, a stem rot disease with an incidence of 3% was found in Baisha city(19°22'18″N,109°16'58″E), Hainan Province, China. In the early stage of the disease, the crown showed chlorotic leaves, followed by defoliation. In later stages, whole tree dieback was observed. The basal tissue of the stem of the diseased tree had white rot, and black-brown basidiocarps were observed about 1 m away from the ground. The basidiocarps surface of fresh was disinfected with 75 % ethanol, the epidermal tissue was removed, and the inner tissue blocks were transferred to PDA medium. After culturing in dark at 28°C for 3 days, a colony with white aerial mycelium was isolated and designated: HNBSMZXS20211011001. The basidiocarp was dark brown, sessile, mostly one-year-old, and the cap is nearly semi-circular, wooden, slightly shiny, with a size of 14.7 to 18.1cm × 9.5 to 10.1 cm. The base is thick (5 to 6.5 cm) and the edge is thin (0.3 to 0.7 cm). The basidiospores are oval, 10.7 to 13.65μm × 6.7 to 9.06μm in size, with a double-layer wall. The outer wall is transparent and the inner wall is light yellow. The basidiospores contain 1~2 oil droplets. The morphological features are consistent with those of Amauroderma subresinosum (Murrill) Corner (Zhang et al., 2000). The basidiocarps and type strain cultures were stored as accessions in the Laboratory of Plant Pathogen Fungus Biology, Hainan University. For pathogenicity tests, sawdust culture medium was used (soft sawdust 82%, wheat bran 15%, glucose 2%, gypsum 1%, mixed with water in proportion, sterilized at 121°C for 40min). The mycelium plug from a fresh culture (d=5mm) was taken from the edge of the colony of type strain, and transferred to the sterilized sawdust medium. When mycelium has colonized the media, it was used to inoculate plants. Media without mycelium was used as a control. Naturally growing seedlings (three year old) of A.mangium were selected from the teaching nurseries of Hainan University (20°6'25''N,110°32'24''E). First, 75 % alcohol was sprayed on the stem of the base of A.mangium for surface disinfection. After the surface was dried, a slight wound (about 4×2cm) was made on the surface with a sterilized scalpel. A inoculated and control sawdust media rods were tightly attached to the wound, moistened with cotton balls soaked in sterile water, and then fixed with plastic wrap, and the outer layer was wrapped with newsprint. Inoculation and controls were replicated three times. Two months after inoculated, the stems of the plants inoculated with the isolated fungus grew white hyphae and showed white rot symptoms, and the leaves became chlorotic and defoliated with complete tree decline in six months, which was consistent with the original symptoms observed. By comparison, white callus had grown on the edge of the stem wounds of the control plants. The same fungus was re-isolated from the inoculated plants and confirmed as A.subresinosum based on the internal transcribed spacer (ITS), the ribosomal large subunit(LSU), and the translation elongation factor 1-α(EF1-α) gene sequence, the fungus was not isolated from control plants thus fulfilling Koch's postulates. The ITS region of r-DNA, the ribosomal large subunit(LSU), the translation elongation factor 1-α gene(EF1-α) were amplified using ITS1/ITS4(White et al. 1990), LR0R/LR5(Hu et al. 2021), EF1-983F/EF1-1567R(Buckley et al. 2005) primers, respectively. The sequences of ITS (OQ674500), LSU (OQ674502) and EF1-α gene (OQ883944) were submitted to GenBank. Through with BLAST, the identities of the ITS, LSU and EF1-α sequences to A.subresinosum (GenBank Accession no. ITS: LC176755; LSU: MK119903 and EF1-α: MK121572) was 99.82%; 99.15% and 99.82%, respectively, the identities were more than 99 %. It was reported that A.subresinosum could infect Casuarina equisetifolia and Areca catechu(Chen et al., 2016; Cheng. 2017; Wu et al., 2019). However, this is the first report of Amauroderma subresinosum causing stem rot of Acacia mangiumin Hainan, China. This report will facilitate field diagnosis and provide scientific reference for further research on the disease.

Discovery of Fusarium pseudograminearum Causing Crown Rot of Winter Wheat in Xinjiang Uygur Autonomous Region, China.

Fusarium pseudograminearum is an important plant pathogen that invades many crops (Zhang et al. 2018). Since it was first discovered in Australia in 1951, F. pseudograminearum has been reported in many countries and regions and caused huge economic losses (Burgess et al. 2001). In 2012, crown rot of wheat caused by F. pseudograminearum was discovered for the first time in Henan Province, China (Li et al. 2012). Wheat (Triticum aestivum L.) is one of the most important food crops in Xinjiang Uygur Autonomous Region (XUAR), with 1.07 million hectares cultivated in 2020. In June 2023, a survey of crown rot disease was carried out in winter wheat cv. Xindong 20 in Hotan area, XUAR, China (80.148907°E, 37.051474°N). About 5% of wheat plants showed symptoms of crown rot such as browning of the stem base and white head. The disease was observed in 85% of wheat fields. In order to identify the pathogens, 36 pieces of diseased stem basal tissue, 0.5 cm in length, were collected and sterilized with 75% alcohol for 30s and 5% NaOCl solution for 2 min, then rinsed three times with sterile water and placed on potato dextrose agar (PDA) medium at 25°C. A total of 27 isolates with consistent morphological characteristics were obtained using single-spore technique (Leslie and Summerell. 2006), and the isolation rate was 75%. The isolates grew rapidly on PDA, produced large numbers of fluffy white hyphae, and pink pigment accumulated in the medium. The isolates were grown on 2% mung bean flour medium and identified by morphological and molecular methods. Macroconidia were abundant, relatively slender, curved to almost straight, commonly two to seven septate, and averaged 22 to 72 × 1.8 to 4.9 μm. Microconidia were not observed. The morphological characters are consistent with Fusarium (Aoki and O'Donnell. 1999). Two isolates (LP-1 and LP-3) were selected for molecular identification. Primers EF1/EF2 (5'-ATGGGTAAGGARGACAAGAC-3'/5'-GGARGTACCAGTSATCATG-3') were used to amplify a portion of the EF-1α gene (O'Donnell et al. 1998). The two 696 bp PCR products were sequenced and submitted to GenBank. The EF-1α gene sequences (GenBank Accession No: PP062794 and PP062795) shared 99.9% identity (695/696) with published F.pseudograminearum sequences (e.g., OP105187, OP105184, OP105179, OP105173). The identification was further confirmed by F. pseudograminearum species-specific PCR primers Fp1-1/Fp1-2 (Aoki and O'Donnell. 1999). The expected PCR products of 518 bp were produced only in F. pseudograminearum. Pathogenicity tests of LP-1 and LP-3 isolates were performed on 7-day-old seedlings of winter wheat cv. Xindong 20 using the drip inoculation method with a 10-μl of a 106 macroconidia ml-1 suspension near the stem base (Xu et al. 2017). The experiment was repeated five times in a 20 to 25°C greenhouse. Control seedlings were treated with sterile water. After 4 weeks, wheat seedling death and crown browning occurred in the inoculated plants with over 90% incidence. No symptoms were observed in the control plants. The pathogen was reisolated from the inoculated plants by the method described above and identified by morphological and PCR amplification using F. pseudograminearum species-specific primers Fp1-1/Fp1-2. No F. pseudograminearum was isolated from the control plants, fulfilling Koch's postulates. To our knowledge, this is the first report of F. pseudograminearum causing crown rot of winter wheat in XUAR of China. Since F. pseudograminearum can cause great damage to wheat, one of the most important food crops in China, necessary measures should be taken to prevent the spread of F. pseudograminearum to other regions.

Green synthesis, structure-activity relationships, in silico molecular docking, and antifungal activities of novel prenylated chalcones.

A series of 16 novel prenylated chalcones (5A-5P) was synthesized by microwave-assisted green synthesis using 5-prenyloxy-2-hydroxyacetophenone and different benzaldehydes. Comparisons were also performed between the microwave and conventional methods in terms of the reaction times and yields of all compounds, where the reaction times in the microwave and conventional methods were 1-4min and 12-48h, respectively. The synthesized compounds were characterized using different spectroscopic techniques, including IR, 1H-NMR, 13C-NMR, and LC-HRMS. The antifungal activities of all compounds were evaluated against Sclerotium rolfsii and Fusarium oxysporum under in vitro conditions and were additionally supported by structure-activity relationship (SAR) and molecular docking studies. Out of the 16 compounds screened, 2'-hydroxy-4-benzyloxy-5'-O-prenylchalcone (5P) showed the highest activity against both S. rolfsii and F. oxysporum, with ED50 of 25.02 and 31.87mg/L, respectively. The molecular docking studies of the prenylated chalcones within the active sites of the EF1α and RPB2 gene sequences and FoCut5a sequence as the respective receptors for S. rolfsii and F. oxysporum revealed the importance of the compounds, where the binding energies of the docked molecules ranged from -38.3538 to -26.6837 kcal/mol for S. rolfsii and -43.400 to -23.839kcal/mol for F. oxysporum. Additional docking parameters showed that these compounds formed stable complexes with the protein molecules.