Translation Elongation Factor 1α Research Articles

First Report of Wilt Caused by Fusarium nanum (FIESC 25) on Mungbean (Vigna radiata) in Pakistan.

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

Phytosanitary problems in elephant garlic (Allium ampeloprasum var. holmense) in the “Val di Chiana” area (Central Italy), and evaluation of potential control strategies

Allium ampeloprasum var. holmense (elephant garlic) is traditionally cultivated in “Val di Chiana”, an area between Umbria and Tuscany regions of Central Italy, under the name “Aglione della Valdichiana”. This product has recently increased in importance, becoming a key economic resource for local farmers. In 2019, phytosanitary problems of elephant garlic cloves ready for transplanting emerged in this cultivation area. Symptom/sign observations and fungal isolations were performed for cloves divided into four components (tunic, basal plate, reserve tissue and shoot) from six farms in the “Val di Chiana” area. Isolates obtained were identified, using partial β-tubulin (BenA) and calmodulin (CaM) or translation elongation factor 1α (tef1α) genes sequences, as belonging to Penicillium [P. allii (95%), P. citrinum (4%), P. brevicompactum (1%)] or Fusarium [F. oxysporum (81%), F. proliferatum (19%)]. Fusarium spp. were mainly associated with clove tunics and basal plates, while Penicillium spp. with basal plates, reserve tissues and shoots. Fungi often also developed from asymptomatic components, but a correlation was found between isolated pathogens and disease symptoms. Pathogenicity and virulence towards elephant garlic cloves were verified for a representative isolate of each identified species, and Penicillium allii was the most virulent. Strategies to control Fusarium and Penicillium spp. on cloves were assessed, including chemicals, a biocontrol agent, surface sterilization and heat treatment. Among these, treatments with Patriot Gold® (active ingredient [a.i.] Trichoderma asperellum TV1, approved in organic farming on crops similar to elephant garlic), or Signum® (a.i. boscalid + pyraclostrobin, approved for Integrated Pest Management systems on crops similar to elephant garlic), were effective in simultaneous reduction of Penicillium spp. and Fusarium spp. Transplanting of asymptomatic cloves combined with the use of the above treatments showed promising effects for pathogens control, and to assist elephant garlic crop establishment.

First Report of Leaf Blight Caused by Botryosphaeria dothidea on Aucuba japonica in Zhejiang Province, China.

Aucuba japonica var. variegata Dombrain is a common evergreen cultivated ornamental in China (Li et al. 2016). In December 2022, severe leaf blight on A. japonica was observed next to the Meishiyuan of Zhejiang Normal University (29°8'4″N, 119°37'54″E) in Jinhua City, Zhejiang Province, China. There were seven plants in the surveyed area, and over 50% of leaves were affected. The early symptoms were small gray spot parts with brown borders on the tip of the leaves. Then the grey parts gradually expanded and became brownish black. In severe cases, the whole leaves became black and blighted. To identify the pathogen, 5 symptomatic leaves were randomly collected from 5 plants and cut into small pieces (5 mm × 5 mm), surface disinfected in 1% sodium hypochlorite solution for 3 min, followed by 75% alcohol for 30 s, then rinsed in sterile distilled water thrice. Tissues were cultured on potato dextrose agar (PDA) and incubated at 28°C for 7 days. Pure cultures were obtained by the single-spore method. Thirteen strains were isolates from the tissues, and nine of them showed similar morphological characteristics. Colonies were white initially, then became gray. The undersides of the colonies became black gradually. Hyaline, fusiform conidia (n = 30) were 17.1 to 24.76 µm (average 20.39 ± 1.906 µm) in length and 5.4 to 6.61 µm (average 6.19 ± 0.434 µm) in width. The DNA of nine isolates were extracted by Ezup Column Bacteria Genomic DNA Purification Kit, and their sequences were identical, so they were named QM1. The internal transcribed spacer (ITS) region, translation elongation factor 1-α (TEF1), and β-tubulin (TUB2) genes were amplified with primer pairs ITS1/ITS4, TEF1-728F/TEF1-986R and βt2a/βt2b (Slippers et al. 2004), respectively. The BLAST analysis indicated that ITS (OR215464), TEF1 (OR243689), and TUB2 (OR243688) of the isolate QM1 were 99 to 100% identical to those of Botryosphaeria dothidea (GenBank accession nos. MH329646 for ITS sequences; OL891702 for TEF1 sequences; MK511445 for TUB2 sequences). In addition, the phylogenetic tree based on sequences from ITS, TEF1 and TUB2 was constructed with MEGA 11 by use of the maximum likelihood method with 1,000 bootstrapping iterations. Based on the multi-locus phylogeny and morphological features, the isolate QM1 was identified as B. dothidea. To test the Koch's postulates, ten leaves from three healthy two- to three-year-old A. japonica plants were surface disinfested with 75% ethanol for 30 s, rinsed with ddH2O three times. The leaves were wounded with a sterile needle and inoculated with 2ml drop of the isolate QM1 conidial suspension (106 spores/mL), with sterile distilled water as a control. All plants were placed in a greenhouse at 28°C, >70% relative humidity and 12 h light/day. The experiment was repeated three times. After 7 days, leaves of the inoculated group showed symptoms similar to those observed on the naturally infected leaves, while leaves of the control group remained asymptomatic. The pathogen was reisolated from inoculated leaves and was confirmed as B. dothidea based on morphological and molecular analyses. It has been reported B. dothidea cause leaf disease in a wide range of hosts in China, such as Camellia oleifera (Hao et al. 2023), Kadsura coccinea (Su et al. 2021). To our knowledge, this is the first report of Botryosphaeria dothidea causing leaf blight on Aucuba japonica in Zhejiang Province of China. B. dothidea are usually secondary invaders and are known to cause diseases in stressed plants. The results further expand the host-range of B. dothidea, and would help to establish control strategy against the disease.

Translation elongation factor-1α is pivotal for plant heat tolerance despite its pronounced heat-induced aggregation

The translation elongation factor 1α (EF1α) protein is a highly conserved G protein that is crucial for protein translation in all eukaryotic organisms. EF1α quickly became insoluble at temperatures 42 °C treatment for 2h in vitro, but generally remained soluble in vivo even after being exposed to temperatures as high as 45 °C for an extended period, which suggests that protective mechanisms exist for keeping EF1α soluble in plant cells under heat stress. EF1α had fast in vivo insolubilization when exposed to 45 °C, resulting in about 40% of the protein aggregating after 9 h. Given its established role in protein translation, heat-induced aggregation is most likely to impact the function of the elongation factor. Overexpression of constitutive mutants in both GTP-bound and GDP-bound forms of EF1α resulted in significantly decreased heat tolerance. These findings provide evidence to support the critical role of EF1α, a thermosensitive protein, in the heat tolerance of plants.

MinION Sequencing of Fungi in Sub-Saharan African Air and a Novel LAMP Assay for Rapid Detection of the Tropical Phytopathogenic Genus Lasiodiplodia.

To date, there have been no DNA-based metabarcoding studies into airborne fungi in tropical Sub-Saharan Africa. In this initial study, 10 air samples were collected onto Vaseline-coated acrylic rods mounted on drones flown at heights of 15-50 meters above ground for 10-15 min at three sites in Ghana. Purified DNA was extracted from air samples, the internal transcribed spacer (ITS) region was amplified using fungal-specific primers, and MinION third-generation amplicon sequencing was undertaken with downstream bioinformatics analyses utilizing GAIA cloud-based software (at genus taxonomic level). Principal coordinate analyses based on Bray-Curtis beta diversity dissimilarity values found no clear evidence for the structuring of fungal air communities, nor were there significant differences in alpha diversity, based on geographic location (east vs. central Ghana), underlying vegetation type (cocoa vs. non-cocoa), or height above ground level (15-23 m vs. 25-50 m), and despite the short flight times (10-15 min), ~90 operational taxonomic units (OTUs) were identified in each sample. In Ghanaian air, fungal assemblages were skewed at the phylum taxonomic level towards the ascomycetes (53.7%) as opposed to basidiomycetes (24.6%); at the class level, the Dothideomycetes were predominant (29.8%) followed by the Agaricomycetes (21.8%). The most common fungal genus in Ghanaian air was cosmopolitan and globally ubiquitous Cladosporium (9.9% of reads). Interestingly, many fungal genera containing economically important phytopathogens of tropical crops were also identified in Ghanaian air, including Corynespora, Fusarium, and Lasiodiplodia. Consequently, a novel loop-mediated isothermal amplification (LAMP) assay, based on translation elongation factor-1α sequences, was developed and tested for rapid, sensitive, and specific detection of the fungal phytopathogenic genus Lasiodiplodia. Potential applications for improved tropical disease management are considered.

First repot of Chaetomium globosum causing leaf spot on Averrhoa carambola in India.

The Star fruit (Averrhoa carambola L.) was globally distributed, particularly in countries like China, India, Indonesia and was renowned for its abundant vitamin, mineral and antioxidant content (Reddy et al., 2023). In early February 2023, leaf spot symptoms were observed on A. carambola at 2 hectare model orchard, College farm, Agricultural College, Aswaraopet (17.252038 latitude, 81.109574 longitude) and Horticulture nurseries of Aswaraopet, Bhadradri Kothagudem (Dist), Telangana, India. In the surveyed fields (February-2023 to February 2024), the disease was prevalent year round, with varying incidence i.e., July to February (35% to 40% with a yield loss of 35%) and from March to June (20% to 30% with a yield loss of 20%). The disease was initiated as small reddish spots, which grew over 8-10 days to 1-5 mm spots with a necrotic center, dark reddish brown margin and a prominent yellow halo around them, within 17 to 20 days, all spots coalesced, resulting in leaf yellowing and defoliation (SF 1). To isolate pathogen, diseased leaf tissues (n=20) (5 × 5 mm) were surface sterilized (70% alcohol (30 s), 1% sodium hypochlorite (30 s) and sterile distilled water (3 × 60 s), inoculated to PDA media and incubated at 26 ± 2°C with 12 hours photoperiod for 72 hours (Chi et al. 2022). The emerging hyphae from the diseased tissues were sub cultured and incubated on PDA at 26 ± 2 °C. Initially, the fungal colonies appeared white, later transitioning to light brown and finally developed into olivaceous grey colour (SF 2A). The ascospores (n=20) were lemon shaped, pointed at both ends, with a length of 10.3 μm (9.1 to 12.1 μm) and width of 8.6 μm (7.2 to 10.2 μm) (SF 2B and 2C). For further identification of the pathogen, four fungal isolates were cultured in potato dextrose broth and genomic DNA was extracted using the CTAB method. Identification of the pathogen was confirmed through amplification and sequencing the Internal Transcribed Spacer region (ITS), Translation Elongation Factor 1-α (TEF1) and RNA polymerase subunit (RPB1) genes. The resulting sequences were deposited in Gen Bank with accession numbers (OR337915, OR337916, OR337893 and OR337892 for ITS, OR669280, OR669281, OR669282, and OR669283 for TEF, and PP092153, PP092154, PP092155 and PP092156 for RPB1). To study pathogenicity of fungus, five isolates of C. globosum were isolated from five A. carambola plants, grown in potato dextrose broth. Spore suspension of 1x106 spores/mL were prepared by adjusting with hemacytometer and were sprayed onto the leaves of healthy, surface sterilized (50% ethanol), 3 months old A. carambola plants and incubated in greenhouse (T: 26°C; RH: 80%). For each of the five isolates, the spore suspension from each individual isolate was inoculated into three plants and three control plants were maintained for each isolate. The experiment was replicated thrice. After a period of 10 to 12 days, symptoms appeared on the inoculated leaves in the form of reddish spots, similar to original symptoms (Alam et al. 2021) (SF 1H). The fungus isolated from the inoculated leaves was morphologically similarities to C. globosum. Notably, C. globosum, a widespread leaf spot pathogen in crops like A. hypogaea, C. sativa and Pomegranate (Chaffin et al. 2020). To our knowledge, this is the first report of A. carambola leaf spot caused by C. globosum in India and worldwide. The result will be helpful for providing a basis for further research on the control of the disease.

Genetic diversity and antagonistic properties of Trichoderma strains from the crop rhizospheres in southern Rajasthan, India

There are fewer studies on Trichoderma diversity in agricultural fields. The rhizosphere of 16 crops was analyzed for Trichoderma species in 7 districts of Rajasthan state of India. Based on DNA sequence of translation elongation factor 1α (tef-1α), and morphological characteristics, 60 isolates were identified as 11 species: Trichoderma brevicompactum, species in Harzianum clade identified as T. afroharzianum, T. inhamatum, T. lentiforme, T. camerunense, T. asperellum, T. asperelloides, T. erinaceum, T. atroviride, T. ghanense, and T. longibrachiatum. T. brevicompactum is the most commonly occurring strain followed by T. afroharzianum. No new species were described in this study. T. lentiforme, showed its first occurrence outside the South American continent. The morphological and cultural characteristics of the major species were observed, described, and illustrated in detail. The isolates were tested for their antagonistic effect against three soilborne plant pathogens fungi: Sclerotium rolfsii, Rhizoctonia solani, and Fusarium verticillioides in plate culture assays. One of the most potent strains was T. afroharzianum BThr29 having a maximum in vitro inhibition of S. rolfsii (76.6%), R. solani (84.8%), and F. verticillioides (85.7%). The potential strain T. afroharzianum BThr29 was also found to be efficient antagonists against soil borne pathogens in in vivo experiment. Such information on crop selectivity, antagonistic properties, and geographic distribution of Trichoderma species will be beneficial for developing efficient Trichoderma-based biocontrol agents.

Wickerhamomyces corioli f.a., sp. nov., a novel yeast species discovered in two mushroom species.

Two yeast strains, designated as 19-39-3 and 19-40-2, obtained from the fruiting bodies of Trametes versicolor and Marasmius siccus collected in Yunwu Mountain Forest Park, PR China, have been identified as representing a novel asexual ascomycetous yeast species. From the results of phylogenetic analyses of the sequences of the D1/D2 domains of the large subunit (LSU) rRNA, small subunit (SSU) rRNA and translation elongation factor 1-α (TEF1) genes, it was determined that these strains represent a member of the genus Wickerhamomyces, with Wickerhamomyces alni and Candida ulmi as the closest relatives. The novel species exhibited 6.6 and 6.7% differences in the D1/D2 domains compared with W. alni and C. ulmi, respectively. Additionally, distinct biochemical and physiological differences were observed between the novel species and its related counterparts. No sexual reproduction was observed in these strains, leading to the proposal of the name Wickerhamomyces corioli f.a., sp. nov. for this newly discovered species.

First report of Fusarium commune Causing Bulb Rot on Lily (Lilium brownii var. Viridulum Baker) in Hunan Province of China.

Lily (Lilium brownii var. Viridulum Baker) is a well-known edible plant with large, white and sweet bulb scales that has important medicinal value (Zhou et al. 2021) and is grown mainly in the Hebei, Shanxi and Henan provinces of China. In May 2021, a case of bulb rot was discovered in a 3.33 hm2 plantation in Huaihua, Hunan Province, affecting 20% of the area (27°59'30″N, 110°32'20″E). The disease is most severe during the rainy season in May and June. In the early stage, irregular brown spots appeared on the lily scales, the necrosis was depressed and gradually enlarges, and in the later stage, the scales were scattered from the base of the disc and slough off. Ten samples were taken randomly from different plants in the plantation area to isolate the pathogens. After washing with sterile water, they were cut into small pieces and sterilised with 3% hydrogen peroxide for 30 s, 75% ethanol for 90 s, rinsed three times with sterile water and dried on sterile filter paper, then placed on a water agar plate and incubated in the dark in a constant temperature incubator at 28℃ for 3 to 5 days. After 2 days, the mycelium at the edge of the colony was transferred to a PDA plate and incubated for 3-5 days at 28°C in the dark to obtain pure fungal isolates. Eighteen purified fungal isolates were obtained, of which sixteen looked like Fusarium (88.9% isolation rate) and three representative isolates (BHBR2, BHBR3 and BHBR5) were selected for further study. The surface of this fungus was white with dense aerial mycelium. Some had an orange centre in the medium. Microconidia were oval in shape and appeared either straight or slightly curved. These microconidia were colourless, had 0-1 septa and measured 3.334 to 14.724 × 2.216 to 5.385 μm (n=100). Macroconidia were predominantly three-septate, crescent-shaped structures that were thin-walled and slightly curved. Cells at the apex and base were similarly curved. Macroconidia measured 17.956 to 32.150 × 2.788 to 4.492 μm (n=100). The mitochondrial small subunit (mtSSU) and translation elongation factor 1-α (TEF1) genes were amplified and sequenced using the NMS1/NMS2 and TEF-R/TEF-F primers to verify the identity of the pathogens (Stewart et al. 2006). The sequences were submitted to GenBank (BHBR2: mtSSU, PP273435; TEF, OR900976; BHBR3: mtSSU, PP277729; TEF, OR900977; BHBR5: mtSSU, PP277728; TEF, OR900978). A concatenated phylogenetic tree of the two genes was constructed and analysis showed that BHBR2, BHBR3 and BHBR5 were significantly clustered with Fusarium commune. Based on the results of morphological identification and phylogenetic tree analysis, the three isolates were identified as Fusarium commune. We carried out pathogenicity tests using two methods, one in which 6 × 6 mm fungal blocks were inoculated on lily (L. brownie var. viridulum Baker) scales and controls inoculated with sterile blocks, and the other in which strain BHBR2 was selected to carry out pathogenicity tests on bulbs of live plants soaked with 50 ml of a 1 × 106 conidial suspension and bulbs of control plants soaked with sterile water, all in three replicates. They were placed in a growth chamber at 28°C and 80% relative humidity, and the scales were moistened with moistened sterile filter paper. After 3 days of rearing treated scales, lesions appeared on lily scales inoculated with mycelial blocks and expanded with time, whereas no lesions appeared on lily scales inoculated with sterile blocks. One month later, whole plants soaked in the spore suspension wilted, while the control plants grew well. The pathogens re-isolated from the diseased tissues had the same morphological characteristics as representative isolates. This confirms Koch's hypothesis. Fusarium commune has been shown to be the most important pathogenic fungus causing root rot in Alfalfa (Medicago sativa) (Yang et al. 2022) and blueberry (Vaccinium uliginosum L.) (Li et al. 2023) in China. To our knowledge, this is the first report of Fusarium commune causing lily bulb rot in the world, which will lay the foundation for future control of lily bulb rot.

First Report of Fusarium Wilt on lily (Lilium 'Tresor') Caused by Fusarium armeniacum in Jiangsu Province, China.

In June 2021, a disease of stem and leaf rot was observed on lily cultivar 'Tresor' with approximately 20% disease incidence in fields at Huaiyin District (119°04'N, 33°63'E) of Huaian County, Jiangsu Province. The roots and bulbs of symptomatic plants were brown and rotten, with sunken lesions. Symptomatic plants showed short, discolored leaves, and eventually lead to stem wilt and death of the whole plants (Fig. 1A and Fig. 3C). To isolate the pathogen, necrotized plant tissues were surface sterilized with 2% sodium hypochlorite for 2 min followed by 70% ethanol for 30 s and rinsed with sterile water. About 4 mm × 4 mm of diseased tissues were placed on potato dextrose agar (PDA) followed by incubation at 25°C in the dark for 5 days. The pure cultures were obtained by the hyphal-tip method. A total of four fungal isolates with similar colony characteristics were recovered. To determine the identity of the four isolated fungal isolates, genomic DNA was extracted using the method previously described (Khan et al. 2021), the sequences of the internal transcribed spacer (ITS), the translation elongation factor 1α (TEF1) and the RNA polymerase II beta subunit (RPB2) genes were analyzed with primers ITS1/ITS4 (White et al. 1990), EF1/ EF2 (O'Donnell et al. 1998), and 5F2/7cR (Reeb et al. 2004), respectively. The three gene sequences of four isolates showed 99.9 %-100% identities. The531 bp (ITS), 699 bp (TEF1), and 900 bp (RPB2) sequences of a representative isolate (JH-37) were deposited in GenBank with acce. nos. OR195729, OR195041 and OR195040, respectively. A phylogenetic tree was constructed using the concatenated three gene sequences of JH-37 and that of the related Fusarium species based on Maximum Likelihood (Fig.2). JH-37 was grouped together with the F. armeniacum strain CBS 485.94 (AB587001, GQ915501, GQ915485), and shared 99.9 % concatenated sequence identity. The three gene sequences of the strain JH-37 shared 100%, 99.85%, 99.89% identity to F. armeniacum strain CBS 485.94 using MEGA 7 software (Kuma et al. 2016) analysis, and with 94%, 95% and 100% coverage by BLAST analysis. The colony of JH-37 on PDA at 25°C for 5 days was white with yellow-brown pigmentation in the center (Fig. 1B-C). From 10-day-old cultures grown on Spezieller Nahrstoffarmer agar (SNA), macroconidia (n = 50) were falcate, slender, curved dorsiventrally, tapering towards both ends, 3 to 4 septate, and measured 24.2 to 50.0 × 2.6 to 4.2 μm. The microconidia (n = 50) were straight or slightly curved, septate 0 to 2, and measured 6.8 to 20.0× 2.1 to 3.7 μm (Fig.1D-F). These morphological characteristics were consistent with Fusarium spp. (Leslie and Summerell 2006). A pathogenicity test of JH-37 was performed on potted lily ('Tresor') under greenhouse conditions. Healthy lily bulbs were selected and one bulb was sown in soil of each pot. Inoculation was performed 60 days after sowing. Bulbs of the lily plants were wounded with needles and inoculated with 5 mL of conidia suspension (1×107 conidia/mL) in the soil around bulb or an equal amount of sterilized water as a control. This experiment had three replicates. After 15 days of inoculation, typical symptoms of bulb rotten, and leaf wilt, similar to the original field symptoms, appeared on the inoculated plants but not on the controls (Fig.3). The same fungus was reisolated from the diseased plants, as identified based on morphology and molecular evidence, which confirmed the Koch's postulate. To our knowledge, this is the first report that F. armeniacum caused Fusarium wilt on Lilium spp. in China. Further, our result could help to develop effective disease management strategies against lily wilt disease.

First Report of Cercospora Leaf Spot Caused by Cercospora cf. flagellaris on Lonicera japonica Thunb. in Henan, China.

Honeysuckle flower (Lonicera japonica Thunb.) is a traditional Chinese medicinal plant. It is perennial and widely cultivated in China, Japan and Korea. From late August to October in 2021 and 2022, leaf spots symptoms were observed on L. japonica in different planting fields in Yuzhou, Yuanyang and Fenqiu districts, Henan province, China. The disease incidence was above 85% which reduce photosynthesis. Early disease symptoms appeared as small, circular to elliptical, brown spots on the leaves and later the lesions (1 to 5 mm × 1 to 4 mm) slowly developed yellow haloes. The different brown lesions seldom merge and form larger irregular lesions. Small fragments (3 to 5 mm) of leave tissue were excised from the lesion margins and surface-sterilized in 3% NaClO for 3 min, followed by three washes with sterile distilled water, and then placed on potato dextrose agar (PDA) and incubated at 25°C in the dark for 5 days. A total number of 8 cultures were obtained and purified by single-spore subcultures on PDA for morphological identification. The colonies on PDA were whitish to gray, with cottony aerial mycelium. Conidiophores were fasciculate, olivaceous brown, straight or geniculate, uniform in width, multiseptate, and ranged from 290 to 700 μm (560 μm on average, n = 20). Conidia were hyaline, slightly curved or straight, needle shaped, truncate at the base, and terminal at the tip, 3 to 17-septate, and measuring 150 to 240 μm (180 μm on average, n = 20). The morphological features were consistent with Cercospora cf. flagellaris Ellis & G. Martin (Groenewald et al. 2013). The genomic DNA was extracted using CTAB method. The nuclear ribosomal internal transcribed spacer region (ITS), portions of the actin (ACT), histone H3 (HIS3), and translation elongation factor 1-α (TEF1) genes were amplified using primers ITS1/ITS4 (Groenewald et al. 2013), ACT-512F/ACT-783R (Carbone and Kohn 1999), CYLH3F/CYLH3R (Crous et al. 2006), and EF1-728F/EF1-986R (Carbone and Kohn 1999). The resulting 537-bp ITS, 226-bp ACT, 410-bp HIS3, and 306-bp TEF1 sequences of isolate JDJ002 were deposited in GenBank (accession nos. OR492367, OR548247, OR548248 and OR548248, respectively). Sequence analysis revealed that ITS, ACT, HIS3 and TEF1α sequences exhibited ≥99% of identity with the ITS (KP896013), ACT(KP895965), HIS3(MK991295) and TEF1 (MN180408) sequences of C. cf. flagellaris, respectively. A pathogenicity test was conducted on healthy of L. japonica leaves. The healthy leaves pricked from L. japonica plants, rinsed in autoclaved distilled water three times and dried with distilled filter paper. Then twelve healthy leave were inoculated with a mycelial plug (0.4 cm diameter) harvested from the periphery of two week-old colony. As negative control, leaves inoculated with PDA medium plugs. Inoculated leaves were covered with plastic bags to maintain high relative humidity and incubated at 25°C in growth chamber. After 7 days, the inoculated leaves showed symptoms identical to those observed in the field under natural conditions, whereas negative control remained symptom-free. Re-isolation of the fungus from lesions on inoculated leaves confirmed that the causal agent was C. cf. flagellaris. Pathogenicity tests were repeated three times by the same methods with the same results. To our knowledge, this is the first report of C. cf. flagellaris except Cercospora rhamni Fack., Alternaria alternata, Corynespora cassiicola or Phomopsis sp. causing leave spots on L. japonica in China.

Endophytic Alternaria and Fusarium species associated to potato plants (Solanum tuberosum L.) in Iran and their capability to produce regulated and emerging mycotoxins

Endophytic fungi live inside virtually every plant species, without causing any apparent disease or damage to the host. Nevertheless, under particular conditions, mutualistic lifestyle of endophytes may change to pathogenic. In this study, the biodiversity of Alternaria and Fusarium species, the two most abundant endophytic fungi isolated from healthy potato plants in two climatically different regions of Iran, Ardebil in the north-west and Kerman in the south-east, was investigated. Seventy-five Fusarium strains and 83 Alternaria strains were molecularly characterized by multi-locus gene sequencing. Alternaria strains were characterized by the sequences of gpd and caM gene fragments and the phylogenetic tree was resolved in 3 well-separated clades. Seventy-three strains were included in the clade A, referred as Alternaria section, 6 strains were included in clade B, referred as Ulocladioides section, and 4 strains were included in clade C, referred as Infectoriae section. Fusarium strains, identified by sequencing the translation elongation factor 1α (tef1), β-tubulin (tub2) and internal transcribed spacer (ITS) genomic regions, were assigned to 13 species, viz. F. brachygibosum, F. clavum, F. equiseti, F. flocciferum, F. incarnatum, F. nirenbergiae, F. nygamai, F. oxysporum, F. proliferatum, F. redolens, F. sambucinum, F. solani and F. thapsinum. Twenty-six selected strains, representative of F. equiseti, F. nirenbergiae, F. oxysporum, F. nygamai, F. proliferatum, and F. sambucinum, were also tested for production of the mycotoxins deoxynivalenol (DON), nivalenol (NIV), diacetoxyscirpenol (DAS), T-2 toxin (T-2), beauvericin (BEA), enniatins (ENNs), fumonisins (FBs), fusaric acid (FA) and moniliformin (MON). None of the tested strains produced trichothecene toxins (DON, NIV, DAS and T-2). Two out of 2 F. equiseti isolates, 1/6 F. oxysporum, 1/3 F. proliferatum, and 1/9 F. nygamai did not produce any of the tested toxins; the rest of strains produced one or more BEA, ENNs, FBs, FA and MON toxins. The most toxigenic strain, F. nygamai ITEM-19012, produced the highest quantities of FBs (7946, 4693 and 4333 μg/g of B1, B2, and B3 respectively), along with the highest quantities of both BEA (4190 μg/g) and MON (538 μg/g). These findings suggest that contamination of potato tubers with mycotoxins in the field or at post-harvest, due to a change in lifestyle of endophytic microflora, should be carefully considered and furtherly investigated.

Chp1 is a dedicated chaperone at the ribosome that safeguards eEF1A biogenesis

Cotranslational protein folding depends on general chaperones that engage highly diverse nascent chains at the ribosomes. Here we discover a dedicated ribosome-associated chaperone, Chp1, that rewires the cotranslational folding machinery to assist in the challenging biogenesis of abundantly expressed eukaryotic translation elongation factor 1A (eEF1A). Our results indicate that during eEF1A synthesis, Chp1 is recruited to the ribosome with the help of the nascent polypeptide-associated complex (NAC), where it safeguards eEF1A biogenesis. Aberrant eEF1A production in the absence of Chp1 triggers instant proteolysis, widespread protein aggregation, activation of Hsf1 stress transcription and compromises cellular fitness. The expression of pathogenic eEF1A2 variants linked to epileptic-dyskinetic encephalopathy is protected by Chp1. Thus, eEF1A is a difficult-to-fold protein that necessitates a biogenesis pathway starting with dedicated folding factor Chp1 at the ribosome to protect the eukaryotic cell from proteostasis collapse.

First Report of Leaf Brown Spot Caused by Diaporthe phoenicicola on Pachira glabra in China.

Pachira glabra is an increasingly important ornamental landscape tree in southern China. In August 2022, brown spots were observed on P. glabra leaves in Xiangtan City, Hunan Province, China (27.932°N, 113.020°E), affecting up to 40% of the 792 trees surveyed. On each diseased tree, nearly 30% leaves had symptoms, with an average severity of 21.2 ± 5.8% (n=100). The disease initially started as small yellow lesions along leaf margins, which later progressed to pale brown to brown with dark brown borders, eventually coalescing into large necrotic areas. Thirty symptomatic leaf samples (2 × 2 mm) were surfaced-sterilized in 75% ethanol for 10 s, 2% NaOCl for 30 s, rinsed in sterile water three times, placed on potato dextrose agar (PDA), and incubated at 25°C for 5 to 7 days in dark. Eight morphologically similar isolates were obtained from diseased leaf samples through single-spore isolation. On PDA, colonies initially appeared white, turning gray, while the reverse developed a pale yellowish hue. Aerial mycelia were white, cottony, and developed visible black pycnidia with oil droplets at maturity. The α-conidia were unicellular, hyaline, aseptate, oval or fusiform, usually with 1 or 2 guttule(s) and rounded at each end. These conidia were 5.3-8.6 × 1.7-2.5 μm (avg. 6.7 × 2.2 μm, n = 100) and present more frequently than β-conidia.The β-conidia were unicellular, hyaline, aseptate, filiform, straight or hamate, eguttulate, 14.6-23.3 × 0.4-1.3 μm (avg. 18.4 × 0.9 μm, n = 30). Morphologically, the fungi were identified as Diaporthe sp. (Udayanga et al. 2014). For molecular identification, the internal transcribed spacer region (ITS), translation elongation factor 1α (EF1-α), calmodulin (CAL), tubulin 2 (TUB2), and histone H3 (HIS3) sequences of all isolates were amplified from genomic DNA, using primers ITS4/ITS5 (White et al. 1990), TEF-2/728F and CALD-38F/CALD-752R (Carbone and Kohn 1999), Bt2a/Bt2b and H3-1a/H3-1b (Glass and Donaldson 1995; Crous et al. 2004), respectively. The GenBank accession numbers for a representative isolate gpg2023-1 were OR533573 (ITS), OR570887 (EF1-α), OR570888 (TUB2), OR570890 (CAL), and OR570889 (HIS3). BLAST results showed that the ITS, EF1-α, TUB2, HIS, and CAL sequences were 99%, 99%, 99%, 99%, and 98% identity, respectively, with those of Diaporthe phoenicicola (GenBank: KC343032.1, KC343758.1, KC344000.1, KC343516.1, and KC343274.1). To confirm the pathogen's identity, phylogenetic analysis using MEGA7.0 based on Maximum Likelihood was constructed. Isolate gpg2023-1 clustered with D. phoenicicola. Based on morphological and molecular data, the fungus was identified as D. phoenicicola. Next, pathogenicity tests were performed three times on one-year-old potted P. glabra plants. For each isolate, twelve healthy leaves on each of three plants were either wounded by a sterile needle or left unwounded, and then sprayed with a conidial suspension (1×106 conidia/ml) for each isolate. Control plants received with sterile water only. Plants were kept in a greenhouse at 25°C, 80% relative humidity, with a 12-h photoperiod. All wounded, inoculated leaves developed brown spot symptoms similar to those observed in the field with six days, while unwounded leaves and control plants remained symptom-free. The fungus was reisolated from all diseased leaves, fulfilling Koch's postulates and proving D. phoenicicola as the causative agent of this brown spot disease on P. glabra. While D. pachirae has been reported to cause leaf spot on P. glabra in Brazil (Milagres et al. 2018), this study marks the first report of D. phoenicicola causing leaf brown spot on P. glabra in China. This finding can help develop control strategies for this disease.

First Report of Leaf Spot Disease on Pineapple Caused by Fusarium solani in China.

Pineapple (Ananas comosus) is an economically important tropical fruit in Guangdong Province, China. The plants were seriously infected with a year-round leaf spot disease. During September to November 2022, the leaf spot disease of pineapple was found in Xuwen city and Zhanjiang city of Guangdong Province. A disease survey of 1 ha revealed that pineapple was affected at an incidence ranging from 30% to 50%. The disease caused economic loss to control plant diseases with chemicals. The initial symptoms were observed after 1 month of planting in October and included yellow spot and developed brown necrotic lesions. The leaves of pineapples showed symptoms of Large brown necrotic lesions appear on leaves especially on the tip of basal leaves. The diseased leaves were collected and surface-disinfested in 1% NaClO for 2-3 m, rinsed with sterilized water, air dried, placed on potato dextrose agar (PDA) medium, and incubated for 3 to 5 days at 28°C. Two isolates (PLF1 and PLF2) were collected and purified using the single-spore method. Colonies developing on PDA were in a circle with abundant white, densely fluffy aerial mycelium, pale to colorless after 3 d and pale orange 5-15 d later. Cultures of the isolates produced macroconidia which were falcate, 1-3-septate, hyaline, 18.2-43.4×4.8-6.8 µm(n=50). Cultures of the isolate also produced large amount of conidia which were hyaline, oblong, no septate, 5.2-10.6×2.7-5.2 µm (n=50). These characteristics were consistent with the description of Fusarium sp. (Chitrampalam et al. 2018). For molecular identification, the genomic DNA of the 2 isolates was extracted. The fragments of internal transcribed spacer (ITS), translation elongation factor 1α (EF1α) and β-tubulin were amplified and sequened using the primer pairs of ITS4/ITS5, EF1/EF2 (O'Donnell et al. 2008) and T1/T2 (O'Donnell et al. 1997). These sequences were deposited in GenBank (OR501466, OR501467 for the ITS; OR499874, OR499875 for elongation factor; OR499876, OR499877 for β-tubulin). Phylogenetic trees were constructed in MAGA 11 using the Maximum likelihood (ML) method based on the concatenated sequences of ITS, EF1α, and Tublin (Figure 1). The 2 isolates were grouped with F. solani A01-1 (GCA_027945525.1) with a bootstrap value of 100 in the phylogenetic tree. The morphology and multi-gene phylogenetic analysis indicated that the new isolates are F. solani. The 2 isolates were selected for pathogenicity tests to fulfill Koch's postulates. Six plants at seven- to ten-leaf stage were inoculated with each isolate separately. Three sites of each leaf were wounded with a sterile needle and covered with a piece of cotton drenched with 200 µl spore suspension (107 spores/ml) from each isolate cultured in PD medium. Leaves inoculated with PD medium served as negative controls. Inoculated plants were placed in an incubator at 28°C, and 80% humidity under a 12-h light/dark cycle for 7 days. After 7 days of incubation, necrotic spots were observed in all the inoculated plants except the negative control. The pathogenicity tests were conducted three times with similar results. The strains were then reisolated from the lesions and found to be Fusarium solani as those of the inoculum. F．ananatum， F. guttiforme and F. subglutinans have been reported to infect all parts of pineapplefusariosis disease (Jacobs et al. 2010; Stępień et al. 2013; Ventura et al. 1993). To our knowledge, this is the first report of fusariosis on Pineapple caused by F. solani. Identification of F. solani as a disease agent on pineapple will assist in disease management for this important fruit tree.

First Report of Charcoal Rot on Soybean Caused by Macrophomina phaseolina in Manitoba, Canada.

Macrophomina phaseolina (Tassi) Goid. is a soilborne necrotrophic fungal pathogen causing charcoal rot on approximately 500 plant species worldwide (Mengistu et al. 2015). Charcoal rot occurs in eastern Canada and many regions of the USA, causing substantial yield losses in soybean [Glycine max (L.) Merr.] (Allen et al. 2017; Bradley et al. 2021; Wrather et al. 2001). However, it has not been reported in soybean in western Canada. Manitoba is the second largest soybean producer in Canada, comprising 31% of total seeded areas with 2.29 M acres in 2017 (Statistics Canada 2022). Still, soybean is a relatively new crop to Manitoba and annual surveys of soybean root diseases began in 2012. In August 2020, randomly selected soybean fields were surveyed for root diseases at 63 different locations in south-central and southwest Manitoba. A total of thirty diseased plants were sampled in a zigzag pattern at three random sites in each field and all samples were brought to the laboratory and rated for disease severity. All plants showed symptoms of root rot, and some samples exhibited wilting with yellowing-brown leaves attached to the stems by the petioles; when the taproot was sectioned longitudinally, black streaking could be observed. In the laboratory, 600 roots from 40 selected fields were processed for pathogen isolation and identification. A 1 cm section from each root was surface-sterilized in a 95% EtOH:5.25% NaOCl solution for 30 sec, rinsed in sterile water for 60 sec, and air-dried on sterilized filter paper in a laminar flow hood. Root tissues with two replicates were placed on potato dextrose agar (PDA) plates amended with streptomycin sulfate (2 mg/mL) and incubated at room temperature. Black microsclerotia were observed in cultures from three different fields and three individual fungal isolates were obtained from each field through isolation of a single microsclerotium and subsequent hyphal tip transfer. The mycelia were initially hyaline and turned gray to dark brown or black, forming numerous microsclerotia ranging in size from 13 to 61 µm long and 12 to 32 µm wide, based on measurements of approximately 100 microsclerotia per isolate using a Zeiss Axio Imager A2 microscope equipped with an AxioCam HRc (Carl Zeiss, Jena, Germany) and AxioVision software. The color of the microsclerotia was jet black and the shape was round to oblong or irregular, as described by Mengistu et al. (2015). Based on morphological characteristics and microscopic examination, three fungal isolates were identified as M. phaseolina (Mengistu et al. 2015). For molecular identification, genomic DNA was extracted from 10 to 14-day old mycelia and microsclerotia of each isolate using a ZymoBIOMICS™ DNA Miniprep Kit (Zymo Research Corp., Irvine, CA, USA) according to the manufacturer's instructions. The internal transcribed spacer (ITS) region, translation elongation factor-1α (TEF-1α), and calmodulin (CAL) genes were amplified using the primer sets ITS1/ITS4 (White et al. 1990), MpTefF/MpTefR, and MpCalF/MpCalR (Santos et al. 2020), respectively, according to the original reaction conditions. Subsequently, PCR products were sequenced at Eurofins Genomics (Louisville, KY, USA). BLASTn analysis in GenBank showed that the nucleotide sequences of these regions of the three isolates (NSRR20-MB-24, NSRR20-MB-34, and NSRR20-MB-40) matched multiple isolates of M. phaseolina with 100% query cover and 100% identity. Sequences were deposited in GenBank for the ITS (OK127887, OK142725, OK128266), TEF-1α (OR363103, OR363104, OR363105), and CAL (OR357627, OR357628, OR357629) regions. In addition, the ITS and TEF-1α sequences of the three novel isolates were further aligned with multiple previously reported isolates of M. phaseolina, M. pseudophaseolina, and M. euphorbiicola (Chen et al. 2013; Machado et al. 2019; Sarr et al. 2014) using Muscle and trimmed (Edgar 2004). Alignments were concatenated to generate a maximum likelihood tree. Once concatenated, sequences were re-aligned. The obtained alignments were employed to construct a phylogenetic tree using the max likelihood method and Tamura-Nei model (Tamura and Nei 1993) with 10,000 bootstrap replicates using MEGA 11 (Tamura et al. 2021). The ITS and TEF-1α analysis indicated that the isolates were grouped in three differentiated clades (Figure 1). Macrophomina phaseolina isolates clustered in the same clade at 98% similarity, with the three novel soybean isolates NSRR20-MB-24, NSRR20-MB-34, and NSRR20-MB-40 grouped closely in the cluster at 98% similarity and identified as M. phaseolina. In contrast, isolates of M. euphorbiicola formed another clade at 87% similarity and M. pseudophaseolina isolates grouped in a clade at 99%. The pathogenicity of the three isolates was evaluated under controlled conditions. Given that no information on charcoal rot resistance in soybean has been reported in Canada, one of the commonly grown varieties in Manitoba, "TH 32004", was selected for the pathogenicity test. Surface-sterilized soybean seeds, which had been pre-germinated for three days, were sown in a sterilized soilless growing mix (Sunshine #5) together with 5 g (approx. 1 × 105 microsclerotia) of macerated 10 to 14-day old inoculum grown on PDA-streptomycin agar medium at room temperature and applied using an inoculum layering technique. For the non-inoculated control, macerated PDA-streptomycin agar without mycelia was used. Twenty plants per treatment were maintained in a walk-in plant growth chamber with a 16 h photoperiod at 25/20 °C ± 1 °C (day/night) and 50% relative humidity. Plants were watered weekly but were subjected to water stress. Symptoms of charcoal rot were observed in the root systems of all inoculated soybean plants after 28 days, while no symptoms were observed in the control plants (Figure S1). There was production of microsclerotia on the roots inoculated with each isolate (data not shown). Three isolates of M. phaseolina were re-isolated from the inoculated plants and found to be identical to the inoculated isolates with respect to morphological characteristics in culture, as well as with respect to the ITS, TEF-1α and CAL DNA sequences. For each isolate and non-inoculated control, five seeds of 'TH 32004' were seeded per pot, and four pots were used for the inoculated and control treatments. The experiment was repeated twice in a randomized complete block design with similar results, fulfilling Koch's postulates. To our knowledge, this is the first report of charcoal rot caused by M. phaseolina on soybean in Manitoba, Canada.

First report of <em>Curvularia dactylocteniicola</em> causing leaf spots on <em>Zea mays</em> and <em>Sorghum</em> in Sri Lanka

The genus Curvularia includes pathogens, endophytes and saprobes associated with a diverse range of plants belonging to the family Poaceae. These species are responsible for diseases on cereal crops such as rice, maize, wheat, millet, sorghum and poaceous weedy hosts. During a field survey of cereal pathogens conducted in Galle, Hambanthota, and Matale districts of Sri Lanka, diseased leaf samples were collected randomly from Zea mays and an indigenous cereal landrace of Sorghum sp. traditionally known as ‘Swayanjatha’. Morphology and molecular phylogeny based on combined nuclear ribosomal internal transcribed spacers 1 and 2 with 5.8S region (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and translation elongation factor 1-α (TEF1) revealed that the causative agent of the leaf spots is Curvularia dactylocteniicola. Results of the pathogenicity tests confirmed cause of the disease on Zea mays and Sorghum sp. To the best of our knowledge, this is the first record of C. dactylocteniicola causing leaf spots on Zea mays and Sorghum sp. in Sri Lanka.

Species distribution and antifungal susceptibility profiles of clinical and environmental Fusarium isolates from Mexico: A multicenter study

ABSTRACT Fusarium spp. has emerged as an opportunistic etiological agent with clinical manifestations varying from localized infections to deep-seated systemic disease. It is also a phytopathogen of economic impact. There are few reports on the species diversity of this genus, and no comprehensive studies on the epidemiology nor the antifungal susceptibility of Fusarium in Mexico. The present multicentric study aims to shed light on the species distribution and antifungal susceptibility patterns of 116 strains of Fusarium isolated from clinical and environmental samples. Isolates were identified by standard phenotypic characteristics and by sequencing of the ITS (internal transcribed spacer), TEF1 (translation elongation factor 1-α), RPB2 (RNA polymerase II core subunit), and/or CAM1 (calmodulin) regions. Susceptibility tests were carried out against 15 antifungals of clinical and agricultural use. Regarding Fusarium distribution, we identified 27 species belonging to eight different species complexes. The most frequently isolated species for both clinical and environmental samples were F. falciforme (34%), F. oxysporum sensu stricto (12%), F. keratoplasticum (8%), and F. solani sensu stricto (8%). All Fusarium isolates showed minimum inhibitory concentrations (MICs) equal to or above the maximum concentration evaluated for fluconazole, 5-fluocytosine, caspofungin, micafungin, and anidulafungin. All isolates had a MIC of ≤16 µg/mL for voriconazole, with a mode of 4 µg/mL. F. verticillioides appeared to be the most susceptible to all antifungals tested.

Methylation of elongation factor 1A by yeast Efm4 or human eEF1A-KMT2 involves a beta-hairpin recognition motif and crosstalks with phosphorylation

Translation elongation factor 1A (eEF1A) is an essential and highly conserved protein required for protein synthesis in eukaryotes. In both Saccharomyces cerevisiae and human, five different methyltransferases methylate specific residues on eEF1A, making eEF1A the eukaryotic protein targeted by the highest number of dedicated methyltransferases after histone H3. eEF1A methyltransferases are highly selective enzymes, only targeting eEF1A and each targeting just one or two specific residues in eEF1A. However, the mechanism of this selectivity remains poorly understood. To reveal how S. cerevisiae elongation factor methyltransferase 4 (Efm4) specifically methylates eEF1A at K316, we have used AlphaFold-Multimer modelling in combination with crosslinking mass spectrometry (XL-MS) and enzyme mutagenesis. We find that a unique beta-hairpin motif, which extends out from the core methyltransferase fold, is important for methylation of eEF1A K316 in vitro. An alanine mutation of a single residue on this beta-hairpin, F212, significantly reduces Efm4 activity in vitro and in yeast cells. We show that the equivalent residue in human eEF1A-KMT2 (METTL10), F220, is also important for its activity towards eEF1A in vitro. We further show that the eEF1A guanine nucleotide exchange factor, eEF1Bα, inhibits Efm4 methylation of eEF1A in vitro, likely due to competitive binding. Lastly, we find that phosphorylation of eEF1A at S314 negatively crosstalks with Efm4-mediated methylation of K316. Our findings demonstrate how protein methyltransferases can be highly selective towards a single residue on a single protein in the cell.

Morphology, phylogeny and pathogenicity of Botryosphaeria dothidea causing leaf blight of Eucalyptus in India

Eucalyptus spp. are widely cultivated in India and worldwide for their versatile uses. Thus, sustainable disease management requires critical knowledge of Eucalyptus pathogens. In December 2022, a leaf blight disease was observed in Eucalyptus plantations in Haryana, northern India. Isolations from the symptomatic leaves yielded Botryosphaeria-like fungal isolates. Megablast homology search and phylogenetic analysis utilizing internal transcribed spacer (ITS), partial translation elongation factor-1α (tef1), and partial β-tubulin (tub2) regions of two isolates confirmed the species as Botryosphaeria dothidea. Leaf inoculations with the two B. dothidea isolates produced disease symptoms on Eucalyptuscamaldulensis clone 2023. Finally, Koch's postulates were established by re-isolation and re-identification of B. dothedia isolates from the inoculated leaves. To the best of our knowledge, this is the first report of B. dothidea causing foliar disease symptoms on Eucalyptus in India. These findings will contribute to developing disease management strategies for Eucalyptus pathogens in northern India.