Β-tubulin Regions Research Articles

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.

Tubulin glycylation controls ciliary motility through modulation of outer-arm dyneins.

Tubulins undergo several kinds of posttranslational modifications (PTMs) including glutamylation and glycylation. The contribution of these PTMs to the motilities of cilia and flagella is still unclear. Here, we investigated the role of tubulin glycylation by examining a novel Chlamydomonas mutant lacking TTLL3, an enzyme responsible for initiating glycylation. Immunostaining of cells and flagella revealed that glycylation is only restricted to the axonemal tubulin composing the outer-doublet but not the central-pair microtubules. Furthermore, the flagellar localization of TTLL3 was found to be dependent on intraflagellar transport. The mutant, ttll3(ex5), completely lacks glycylation and consequently exhibits slower swimming velocity compared with the wild-type strain. By combining the ttll3(ex5) mutation with multiple axonemal dynein-deficient mutants, we found that the lack of glycylation does not affect the motility of the outer-arm dynein lacking mutations. Sliding disintegration assay using isolated axonemes revealed that the lack of glycylation decreases microtubule sliding velocity in the normal axoneme but not in the axoneme lacking the outerarm dyneins. Based on our recent study that glycylation occurs exclusively on β-tubulin in Chlamydomonas, these findings suggest that tubulin glycylation controls flagellar motility through modulating outer-arm dyneins, presumably by neutralizing the negative charges of glutamate residues at the C-terminus region of β-tubulin.

Discovery of Botryosphaeria eucalypti sp. nov. from blighted Eucalyptus leaves in India

Eucalyptus spp. are undoubtedly one of the most favored plantation trees globally. Accurately identifying Eucalyptus pathogens is therefore crucial for timely disease prevention and control. Recently, symptoms of a leaf blight disease were observed on Eucalyptus trees in plantations at Jhajjar and Karnal in the state of Haryana, northern India. Asexual morphs resembling the features of the Botryosphaeriaceae were consistently isolated from the symptomatic leaves. Morphological features coupled with DNA sequence analysis confirmed a novel species, which is described and illustrated here as Botryosphaeria eucalypti sp. nov. Conidia of the new taxon are longer and wider than those of its phylogenetic neighbors. A distinct phylogenetic position for the new taxon was established through combined analysis of the internal transcribed spacer (ITS), partial translation elongation factor-1α (tef1) and partial β-tubulin (tub2) regions. Recombination analysis provided additional support for the new species hypothesis. The pathogenicity of the novel species was proved on Eucalyptus leaves, and Koch's postulates were fulfilled. The discovery of new Botryosphaeria species is important because it will help in understanding the species diversity, host range, possible threats and disease control in the long run.

Neofusicoccum mangiferae causing leaf spot disease of Eucalyptus: First report from India

Fungal diseases are a serious threat to Eucalyptus spp., which are cultivated worldwide for pulp and paper. Robust and timely detection of Eucalyptus pathogens is thus a crucial step in disease prevention and control. In 2022, leaf spots were observed on Eucalyptus trees in Karnal, northern India. Fungal isolates displaying morphological characteristics similar to Neofusicoccum mangiferae were consistently recovered from the infected leaves. The morphological identification was validated by phylogenetic analysis employing internal transcribed spacer (ITS), partial translation elongation factor-1α (tef1) and partial β-tubulin (tub2) regions. Finally, Koch's postulates for N. mangiferae as a foliar pathogen of Eucalyptus were fulfilled. This is the first confirmed report of N. mangiferae affecting Eucalyptus trees in India.

First Report of Shoot Blight of Grapevine Caused by Sclerotinia sclerotiorum in Illes Balears, Mallorca, Spain.

In 2021, grapevines (Vitis vinifera L.) cv. Callet growing in a commercial vineyard located at Pollença (northeast of the island of Majorca, Spain) showed severe symptoms of shoot blight during spring and early summer, with an incidence of 70%. Symptoms consisted of elongated cankered-like lesions, surrounded by water-soaked darker tissues, that developed at the base or around the middle nodes of the shoot. For fungal isolation, shoot samples with lesions were collected, surface disinfected with 2% NaCl for 90s, rinsed twice with deionized water and placed in Petri plates containing potato dextrose agar (PDA). The plates were incubated at 25°C under 12 h light-darkness for 6 days. Isolations consistently yielded on kind of fungal colonies that produced white mycelium and black spherical to elongated sclerotia (2 to 10 mm in diameter). Morphological characterization was consistent with the description of Sclerotinia sclerotiorum (Lib.) de Bary (Bolton et al. 2006). Three isolates (UIB 118-1, UIB 118-26, and UIB 129-41) were preserved and deposited in the Culture Collection of Microbiology-Faculty of Sciences, University of Balearic Islands, Spain. Genomic DNA was extracted from isolates UIB 118-26 and UIB 129-41 using the EZNA Miniprep Kit (Omega Bio-Tek, Norcross, GA). The internal transcribed spacer (ITS) region of ribosomal DNA, β-tubulin (BTUB) and calmodulin (CAL) gene regions were amplified using ITS1F-ITS4 (Gardes and Bruns, 1996; White et al. 1990), Bt-2a/Bt-2b (Glass and Donaldson 1995) and CAL228F/CAL737R (Carbone and Kohn 1999) primer sets, respectively. Amplicons were sequenced and deposited in GenBank with accession numbers MZ604647 and MZ604648 for ITS, OK634402 and OK634403 for BTUB and OK634404 and OK634405 for CAL. BLASTn search showed that isolates were >99 % (ITS, BTUB and CAL) identical to S. sclerotiorum GenBank accession no. KF859933, CP017815 and KF871381, respectively. Pathogenicity tests were conducted using eight one-year old grapevines cv. Cabernet Sauvignon. Old and new green shoots were inoculated by inserting a 6-mm plug of mycelium taken from actively growing cultures on PDA into cuts made at the base and at the distal part of each shoot with a sterile scalpel with a total of eight inoculation points per plant. Inoculated wounds were sealed with Parafilm tape to avoid rapid dehydration. Inoculated plants and an equal number of wounded but non-inoculated plants (negative controls) were maintained at 25 ± 1°C for 48 h in plastic containers to ensure a high relative humidity (>90%). After 5 days, the infection girdled and rotted the green new shoots, whereas the older partially lignified shoots developed a localized long brown lesion that reached 16 cm in length. Due to the rotting of the basal part of the petiole, leaves turned gray, wilted, and died, easily detaching from the stem. In advanced stages of the disease, 7 days after infection, branches died and fell with the leaves remained attached (Fig 1 A, B). Reisolations from diseased shoots were successfully performed on PDA to fulfill Koch's postulates. S. slerotiorum was previously reported on grapevine causing shoot blight in Chile (Latorre and Guerrero, 2001), Korea (Jong-Han et al. 2009), California-USA (Boland and Hall, 1994) and Australia (Hall et al. 2002). AlsoS. sclerotiorum was reported among the endophytic mycobiota associated with Vitis vinifera in the Iberian Peninsula (Gonzalez and Tello, 2011) but not as a pathogen causing visible symptoms on that crop. So, this is the first report of the occurrence of S. slerotiorum as a pathogen of grapevines in Spain causing symptoms of canker and shoot blight. This finding highlights a potential risk of this fungal disease for the wine industry in the Mediterranean region and specially for Spain, the country with the largest acreage devoted to grapevines. Although chemical and biological are suitable control strategies, disease management is difficult as sclerotia of Sclerotinia can remain in the soil for up to eight years (Adams and Ayears, 1979), and preventive surveys are greatly recommended as an important epidemiological tool to monitor the epidemiology of disease and identify potential outbreaks of this new pathogen on grapevine in Spain.

First Report of Black Leaf Spot Caused by Epicoccum latusicollum on Chinese Yam in China.

Chinese yam (Dioscorea polystachya Turczaninow cv. Tiegun), which belongs to the family Dioscoreaceae, is widely cultivated throughout China due to its high economic and medicinal value. In June 2023, black leaf spots on Chinese yam (cv. Purple 1) were observed in Nanchang city (28.45° N, 115.49° E) of Jiangxi province, southeastern China. The incidence of the disease ranged between 70 and 85% of plants, and up to 30% of the leaves per plant were affected in the field over a 2-week period of study. Infected foliage displayed brown necrotic lesions, elliptical or irregular, with yellow halo at the edge of the lesion (0.5 to 3 cm diam.). To identify the causal agent, 32 symptomatic leaves of eight symptomatic plants were collected. Small pieces from the margin of necrotic leaf tissue (about 3 x 3 mm) were surface sterilized in 75% ethanol for 30 s followed in 0.1% HgCl2 for 1 min, and washed three times with ddH2O. Then, the pieces were transferred onto potato dextrose agar (PDA) plates and incubated at 26°C for 3 days with a 12-h light-dark cycle. From the 32 isolates, 21 exhibited similar morphology after hyphal tipping resulting in an isolation frequency of 65.6%. Colonies on PDA were initially white aerial hyphae but became grayish with age, and a reddish orange pigment on the underside. After 16 days of incubation, pycnidia were observed, which were dark, spherical or flat spherical, and 64.1 to 172.5 µm (n = 25) in diameter. Conidia were ellipsoidal, aseptate, hyaline, and 4.1 to 5.6 × 1.8 to 2.7 µm (n = 80). In addition, a blackish green discoloration was produced on malt extract agar (MEA) using the NaOH spot test. The isolates were tentatively identified as Epicoccum spp. based on morphological characteristics (Chen et al. 2017). Isolate AYZ-1 was randomly selected for identification and pathogenicity testing. Genomic DNA of the isolate (AYZ-1) was extracted and amplified by polymerase chain reaction (PCR) using ITS1/ITS4 for the internal transcribed spacer (ITS) region (White et al. 1990), Btub2Fd/Btub4Rd for the β-tubulin (TUB) region (Woudenberg et al. 2009), LROR/LR7 for the large ribosomal RNA gene (LSU) region (Rehner and Samuels 1994), and RPB2-5F2/fRPB2-7cR for RNA polymerase II second largest subunit (RPB2) region(Liu et al. 1999), respectively. The concatenated sequences (GenBank Accession No. OR574165, OR567827, OR574166, OR567828, respectively) shared 99.8 to 100% identity withEpicoccum latusicollum(OP788080, MN329871, OR428532, and OL422485, respectively). A neighbor-joining phylogenetic tree was generated based on the concatenated sequences in MEGA7, placed isolate (AYZ-1) within E. latusicollum. To fulfill Koch's postulates, healthy leaflets from three one-year-old Chinese yam (cv. Purple 1) were used as inoculation materials, using isolate AYZ-1. Two sites of each leaf were wounded with a sterile needle and covered with a piece of cotton drenched with 200 µL spore suspension (106 conidia/mL) on the left sides, while sterilized water served as the control on the right sides of leaves. All inoculated leaves were covered with clear polyethylene bags for 24 h. Plants were grown outdoors at a daily average temperature of 26°C with relative humidity over 45%. After 7 days of incubation, the leaves showed the same symptoms as the original diseased leaves. The E.latusicollumisolate was re-isolated from diseased leaves and confirmed by morphology and sequencing analysis, fulfilling Koch's postulates. E.latusicollumhas beenpreviously reportedtocauseblack root on yam inChina'ssouth-westernprovince ofSichuan(Han et al. 2019). Meanwhile,leafspothave been reportedon many plants by this genus, such astobacco(Guo et al. 2020) andbanana(Liu et al. 2023). According to our knowledge, this is thefirstreportofE.latusicollumcausingblack leafspoton Chinese yam inChina. This finding will provide an important reference for understanding the biology of E. latusicollum and the distribution of the disease, but more research is needed to determine if management is warranted.

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.

First Report of Lasiodiplodia pseudotheobromae Causing Necrosis and Canker Disease on Pear Tree in China.

Pear ispopularamongpeople, which is an important pillar industry in China. In March of 2023, dark brown necrotic lesions were discovered on the trunks of Pyrus pyrofolia cv. Osmanthus pear in orchard, Liuzhou City, Guangxi Zhuang Autonomous Region. In August, field investigation and sample collection were conducted in orchard. Forty pear trees were selected for symptomatic observation, which of 21 had lesions ranging from 10 to 24 per tree, and 19 with 1 to 8 lesions, respectively. To isolate the pathogen, small tissue pieces of 3 diseased pear trunk samples were disinfected with 75% ethanol for 1 minute, rinsed with sterile water, and dried with filter paper. The tissue pieces were placed on potato dextrose agar (PDA) plates and cultured in a dark incubator at 25℃. Six isolates with the similar morphology were obtained. One of the six isolates was randomly selected as the representative strain and named as GX-3. Mycelium grows with an average rate of 4.26 cm/d. The hypha is highly aerial, and is initially white and then turns black. Subsequently, pycnidia formed and secreted black mucus on the PDA medium after 28 days. The immature conidia were ellipsoid, colorless, hyaline, and unicellular, mostly becoming brown bicellular with longitudinal stripes at maturity. The conidial size was 22.5 to 32.6×12.1 to 19.7μm, and the average size was 28.4±2.3×16.7±2.0 μm (n=50), respectively. GX-3 colony morphology was consistent with that of Lasiodiplodia pseudotheobromae (Alves et al.2008). For molecular identification, the internal transcribed spacer of rDNA (ITS), translation elongation factor 1-α (TEF1-α), and β-tubulin regions were amplified using the primers ITS1/4, EF1-728F/986R, and Bt2a/Bt2b, respectively (White et al.1990; Carbone and Kohn 1999; Glass and Donaldson 1995). The obtained sequences of GX-3 were deposited in NCBI with Accession numbers OR655421, OR661231, and OR661230, respectively. The sequences of ITS, TEF1-α, and β-tubulin from GX-3 are 99.44%、99.67% and 99.78% identities with those of L. pseudotheobromae CBS 447.62, respectively. The phylogenetic analysis was performed by maximum likelihood method, revealing that GX-3 is closely clustered with the isolates of L. pseudotheobromae. Therefore, the GX-3 strain was identified as L. pseudotheobromae. GX-3wasfurtheranalyzedforitspathogenicityonpear. Firstly, the GX-3 mycelium plugs and spraying spore suspension with the concentration of 1×107 conidia/ml were applied on the stems of 4-month-old healthy birch-leaf pear (Pyrus betulifolia Bunge) potted seedlings by acupuncture needle method, meanwhile PDA and sterile waterwere usedascontrols. After 3 days of inoculation, stem surface of the birch-leaf pear exhibited dark brown lesions with slight surface depression, obvious dryness, and canker symptoms, while the control treatment showed no symptoms. The GX-3 was also inoculated on in vitro branches of 'Hosui', 'Hongxiangsu', 'Bodoqing' and 'Xuehua', showing dark brown canker lesions. The same pathogen can be successfully isolated from diseased stems and branches but not from the controls, which accomplishes Koch's postulates. L. pseudotheobromae has been widely reported that it can cause rot and canker on apple, walnut, hackberry, and so on (Xue et al. 2019; Wang et al. 2023; Liang et al. 2020). This is the first report of necrosis and canker disease caused by L. pseudotheobromae on pear in China, which is a potential threat to pear industry.

Molecular Characterization of Fungal Isolates of Locally Processed Rice from Five Agro-Ecological Zones of Nigeria

The purpose of this study was to characterize fungal isolates contaminating locally processed rice in Nigeria and to assess the genetic similarity in the fungi isolates. Fungal species were characterized by amplification of a segment of the β-tubulin gene using Bt2aF (5’-GGTAACCAAATCGGTGCTGCTTTC-3’) and Bt2bR (5’ACCCTCAGTGT GT GACCCTTGGC-3’) primers and phylogenetic relationship established using the Maximum Likelihood method. Amplification of between 450 and 550bp of the β-tubulin region of the genomic DNA of the fungal isolates was obtained from thirty-eight samples (89%). Members of Aspergillus section flavi and section nigri groups were differentiated as A. flavus, A. parasiticus, A. tubigensis, A. sydowii and A. niger respectively. Molecular method identified Paecilomycesformosus with 99% similarity index while all the morphologically identified Penicillium species were identified to genus level as P. formosus, P. steckii and P. citrinum with a percentage similarity ranging from 95%-100%. Collectotrichum fruticola and Talaromyces flavus from SGS and NGS zone respectively were documented for the first time in the locally processed rice with 100% and 89% similarity index respectively. All the Aspergillus species were closely related to one another and narrowly diverged from the Penicillium species. Aspergilla and Penicillia were the predominant genera identified and the isolates were genetically similar irrespective of their agroecological zones.

First Report of Fusarium acuminatum Causing Leaf Spot on Schisandra chinensis in China.

Schisandra chinensis (Turcz.) Baill. is a popular and widely cultivated medicinal herb in China, which has rich nutritional value and medicinal effect. In August 2022, leaves with oval and irregularly circular light brown spots from 2 to 10 mm wide with white centers were found on Schisandra chinensis growing in Fusong district (127°28'E, 42°33'N) of Jilin, China. The symptoms were observed in 20% of the plants of a 2 ha-1 field of Schisandra chinensis. About 50% of the leaf areas were affected. As the disease developed, the lesions grew larger and developed necrotic centers. Leaves with light brown spot symptoms from five plants were collected from the field. Five leaf pieces (3 to 5 mm2) were excised from lesion margins, surface sterilized based on Ju et al. (Ju et al. 2021), and incubated on potato dextrose agar (PDA) at 25°C. Six single spores were isolated from five independently infected isolates for pure culture using the single spore isolation technique (Zhang. 2003). Representative single spore isolate (ZWWZH) was selected from pure cultures for further culture. After 5 days, fluffy white aerial mycelium with pink pigmentation on the underside of the colony were observed on PDA. Mycelia became pinkish-brown as the culture aged. Microscopic observations showed the presence of elongated or pointed, and thick-walled macroconidia (n = 50), predominantly three septate, 3.40 to 7.50 × 40.34 to 61.29 μm were observed. Chlamydospores formed in chains within or on top of the mycelium. The primers ITS1/ITS4 (White et al. 1990) and Bt-2a/Bt-2b (Robideau et al., 2011) were used to amplify the internal transcribed spacer (ITS) rDNA and β-tubulin (TUB2) region, respectively. The obtained sequences were submitted to GenBank under accession numbers for OQ629789 (ITS) and OQ803521(TUB2). BLASTn analysis of both ITS sequence and TUB2 sequence, revealed 100% and 99.92% sequence identity with F. acuminatum MT566456, MT560377 and KJ396328, respectively. The pathogen was identified as F. acuminatum based on morphological and molecular data. Pathogenicity tests were carried out in the greenhouse. Select five healthy Schisandra chinensis seedlings, each with each healthy leaf surfaces inoculated a 1 × 106 spores/mL solution, 3 wells on one side, 10 µL per well. Sterile ddH2O was used in the control experiment. The inoculated seedlings were incubated at 25°C with a relative humidity of 65 to 70% in a greenhouse. Four days after inoculation, all inoculated leaves exhibited the same symptoms as observed in the field, while the controls showed no symptoms. The experiment was repeated three more times with similar results. The re-isolated fungi from the inoculated plants had the same morphology and DNA sequences as the original isolate (ZWWZH) obtained from the field samples, completing Koch's postulates. To our knowledge, this is the first report of F. acuminatum causing leaf spot on Schisandra chinensis in China. F. acuminatum has seriously affected the quality of Schisandra chinensis production. The identification of leaf spot caused by F. acuminatum will enable farmers to identify practices to minimize disease on this important crop.

First Report of Charcoal Rot Caused by Macrophomina phaseolina on Stevia rebaudiana in Arizona, USA.

Stevia (Stevia rebaudiana Bertoni) is an important medicinal crop grown worldwide. Leaves of stevia contain a non-caloric sweetener, stevioside, which is used as a substitute to artificial sweeteners. In August 2022, symptoms of chlorosis, wilting, and root rot were observed in about 30 % of stevia plants growing at the Agricultural Station at Yuma Agricultural Center, Yuma, AZ, USA (32.7125° N, 114.7067° W). Infected plants initially showed chlorosis and wilting, and the plants eventually died with foliage remaining intact to the plant. Cross sections of the crown tissue of affected stevia plants showed necrotic tissue and a dark brown discoloration in areas of the vascular and cortical tissues. Dark brown microsclerotia were observed on stem bases and necrotic roots of the infected plants. Five symptomatic plants were sampled to isolate the pathogen. Root and crown tissues (0.5 to 1 cm) were surface disinfested with 1% sodium hypochlorite for 2 min, rinsed three times with sterile water, and plated onto potato dextrose agar (PDA). All the five isolates displayed rapid mycelial growth on PDA at 28°C with a 12-h photoperiod. The mycelia were initially hyaline and turned from gray to black after 7 days. Masses of dark spherical to oblong microsclerotia were observed after 3 days on PDA, measuring an average of 75 µm width × 114 µm length (n=30). For molecular identification, genomic DNA was extracted from mycelia and microsclerotia of a representative isolate (Yuma) using the DNeasy Plant Pro kit (Qiagen, Hilden, Germany). The internal transcribed spacer (ITS), translation elongation factor-1α (TEF-1α), calmodulin (CAL), and β-tubulin (β-TUB) regions were amplified using the primer sets, ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), MpCalF/MpCalR (Santos et al. 2020), and T1/T22 (O'Donnell and Cigelink 1997), respectively. A BLAST search of sequences revealed 98.7 to 100% identity to Macrophomina phaseolina sequences (MK757624, KT261797, MK447823, MK447918). Both morphological and molecular characteristics confirmed the fungus as M. phaseolina (Holliday and Punithaligam 1970). Sequences were submitted in the GenBank under accession numbers OP599770 (ITS), OP690156 (TEF-1α), OP612814 (CAL), and OP690157 (β-TUB). Pathogenicity assay was performed on 9-week-old stevia plants (var. SW2267), grown in 4-inch planters in the greenhouse. The inoculum was made from a 14-day-old culture of M. phaseolina grown in conical flasks (250 ml) in potato dextrose broth at 28°C. Mycelial mats of the fungus were blended in 250 ml of sterile distilled water, filtered through four layers of cheesecloth, and then calibrated to 105 microsclerotia/ml using a hemocytometer. Twenty healthy plants were inoculated by soil drenching 50 ml of the inoculum per pot. Soil drenching using sterile distilled water was done on 5 non-inoculated control plants. Plants were maintained in the greenhouse at 28 ± 3°C with 12 h photoperiod. After 6 weeks, necrosis at the base of petioles and chlorosis of the leaves, followed by wilting were noticed on all 20 inoculated plants, whereas all the 5 control plants remained healthy. The fungus was reisolated and identified as M. phaseolina based on the morphology and sequences of ITS, TEF-1α, CAL and β-TUB regions. Although M. phaseolina has been reported earlier on stevia in NC, USA (Koehler and Shew 2018), this is a first report from AZ, USA. M. phaseolina is known to be favored by high soil temperatures (Zveibil et al. 2011), thus represents a potential threat to stevia production in AZ, USA in coming years.

Molecular characterization of gliotoxin-producing Aspergillus fumigatus in dairy cattle feed.

Several strains of Aspergillus fumigatus produce mycotoxins that affect the health and productivity of dairy cattle, and their presence in dairy cattle feed is a serious concern. This study aimed to determine the densities of A. fumigatus and gliotoxin in commercial dairy feed. More than 60 dairy feed samples were examined for fungal contamination, specifically for A. fumigatus, using phenotypic approaches and DNA sequencing of the internal transcribed spacer (ITS) and β-tubulin regions. Thin-layer chromatography and high-performance liquid chromatography (HPLC) were used to assess gliotoxin production in A. fumigatus. Real-time polymerase chain reaction (RT-PCR) was used to investigate the expression of gliZ, which was responsible for gliotoxin production. High-performance liquid chromatography was used to detect gliotoxin in feed samples. Aspergillus was the most commonly identified genus (68.3%). Aspergillus fumigatus was isolated from 18.3% of dairy feed samples. Only four of the 11 A. fumigatus isolates yielded detectable gliotoxins by HPLC. In total, 7/11 (43.7%) feed samples tested had gliotoxin contamination above the threshold known to induce immunosuppressive and apoptotic effects in vitro. The HPLC-based classification of isolates as high, moderate, or non-producers of gliotoxin was confirmed by RT-PCR, and the evaluation of gliZ expression levels corroborated this classification. The identification of A. fumigatus from animal feed greatly depended on ITS and β-tubulin sequencing. Significant concentrations of gliotoxin were found in dairy cattle feed, and its presence may affect dairy cow productivity and health. Furthermore, workers face contamination risks when handling and storing animal feed.

First report of Lasiodiplodia iraniensis causing leaf spot disease of Eucalyptus in India

Eucalyptus species are highly valuable source for wood-based paper and pulp industries in India and worldwide. Therefore, accurate identification of pathogens affecting Eucalyptus is of critical importance. In March 2022, symptoms of a leaf spot disease were observed in Eucalyptus plantations in northern India. Morphological characteristics coupled with phylogenetic analysis of combined internal transcribed spacer (ITS), partial translation elongation factor-1α (tef1), and partial β-tubulin (tub2) regions identified the associated species as Lasiodiplodia iraniensis. The pathogenicity of L. iraniensis was confirmed on detached Eucalyptus leaves and reconfirmed in glasshouse inoculation experiments. Finally, re-isolation of the pathogen from the inoculated leaves fulfilled the Koch's postulates. This study is the first report of pathogenic L. iraniensis on Eucalyptus in India. Further studies on the diversity of this pathogen and identification of disease resistant Eucalyptus germplasm are required.

First Report of Colletotrichum sansevieriae Causing Anthracnose of Snake Plant (Dracaena trifasciata) in Ohio and its Draft Genome.

Dracaena trifasciata (Prain) Mabb. is a popular houseplant in the United States. In September 2021, two diseased samples from two Ohio homeowners were received by the Ornamental Pathology Laboratory at The Ohio State University. Each sample included one or two detached leaves displaying circular gray water-soaked lesions scattered throughout the lamina and blighted areas with concentric rings bearing brown to black acervuli. Lesions covered between 25 and 50% of the leaf surface. Isolations were made by excising small portions of leaf tissue from the margin of the lesions, surface-disinfesting in 10% bleach for 45 s, rinsing in sterile water, and plating on potato dextrose agar (PDA). Plates were incubated at 23°C for one week. Two representative isolates, one per sample (FPH2021-5 and -6), were obtained by transferring hyphal tips to fresh PDA plates. Mycelia of both isolates were aerial, cottony, grayish-white, producing spores in a gelatinous orange matrix, and appeared gray to olivaceous-gray on the plate underside. Conidia produced by both isolates were cylindrical, single-celled, hyaline, measuring 12.02 to 18.11 (15.51) × 5.03 to 7.29 (6.14) μm (FPH2021-5; n=50) and 15.58 to 20.90 (18.39) × 5.63 to 8.27 (7.05) μm (FPH2021-6; n=50). Appressoria were globose to subglobose, single-celled, dark brown to sepia, measuring 6.62 to 13.98 (8.97) × 5.05 to 6.58 (6.58) μm (FPH2021-5; n=50), and 6.54 to 11.32 (8.63) × 4.54 to 8.94 (7.09) μm (FPH2021-6; n=50). Genomic DNA (gDNA) samples were extracted from both isolates and the internal transcribed spacer (ITS) region was amplified using primers ITS1F/ITS4 (Gardes and Bruns, 1993; White et al. 1990). GenBank BLAST sequence analysis resulted in 99.83% (FPH2021-5; GenBank Acc. No. OP410918.1) and 100% (FPH2021-6; OP410917.1) identity with 100% query coverage to the type strain of Colletotrichum sansevieriae Miho Nakam. & Ohzono MAFF239721 or Sa-1-2 (NR_152313.1; Nakamura et al. 2006). Whole genome sequencing was conducted for FPH2021-6 and the assembly was deposited in GenBank (JAOQIF000000000.1). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-tubulin (β-tub) regions were either extracted from the genome of FPH2021-6 (OP414603.1 and OP414601.1, respectively) or amplified from FPH2021-5 gDNA using primers GDF/GDR (OP414604.1) and Bt-2b/T1 (OP414602.1), respectively (Templeton et al. 1992; Glass and Donaldson 1995; O'Donnell and Cigelnik 1997). A multilocus partitioned analysis (Chernomor et al. 2016) based on concatenated sequences of ITS, GAPDH, and β-tub using ModelFinder (Kalyaanamoorthy et al. 2017) was performed to build a maximum likelihood tree (IQ-TREE v2.0.3; Nguyen et al. 2015), suggesting that these two isolates are phylogenetically closer to the type strain from Japan than to a previously reported isolate 1047 from Florida (Palmateer et al. 2012). To fulfill Koch's postulates, two parallel leaf sections from one 10-inch D. trifasciata 'Laurentii' plant maintained in a 1.3-liter container were selected. Three wounds were made in each section using a sterile syringe needle. A 10-µl drop of either a 1×106 conidia/ml suspension of isolate FPH2021-6 or sterile water was placed on each wound. The plant was covered with a plastic bag for two days post-inoculation (DPI) and maintained in a greenhouse at 25°C with a 12- h photoperiod. The experiment was conducted twice. Grayish water-soaked lesions, acervuli, and leaf blight were observed on the inoculated sections 3, 10, and 14 DPI, respectively, while no symptoms appeared on the sections treated with sterile water. C. sansevieriae was re-isolated from the lesions and confirmed to be identical to the original isolate based on ITS sequencing and morphological examinations. To the best of our knowledge, this is the first report of C. sansevieriae on D. trifasciata in Ohio and the first genome draft of an isolate from the United States. Availability of whole-genome sequence data is paramount for resolving species identification in this highly diverse fungal genus, and a powerful tool to conduct comparative genomic analyses in the future.

Epicoccum sorghinum as leaf spot disease-causing pathogen in ginger (Zingiber officinale Rosc.) in China

Ginger is widely cultivated in China, and it is an indispensable spice and condiment in our daily life. In October 2020, ginger plants exhibiting leaf spot symptoms were observed in many agricultural fields in the Shandong province. In this research, the pathogen causing these symptoms was successfully isolated from symptomatic ginger leaves. The fungal isolates were identified as Epicoccum sorghinum using morphological characteristics and multigene phylogenetic analysis of internal transcribed spacer region of ribosomal DNA (ITS), 28S nrDNA (LSU), and β-tubulin (tub2). Pathogenicity tests showed that the symptoms caused by the pathogen were similar to those on naturally infected plants in the field, confirming that E. sorghinum was the causal agent of ginger leaf spot. To our knowledge, this is the first report of E. sorghinum as the pathogen of leaf spot on ginger in China. These findings provide essential information for formulating effective management strategies to curb the disease spread.

Characterization of Three Fusarium spp. Causing Wilt Disease of Cannabis sativa L. in Korea

In July 2021, wilting symptoms were observed in adult and seedling hemp (Cannabis sativa L. cv. Cherry Blossom) plants grown in a greenhouse. As the disease progressed, yellowing and wilting symptoms on the leaves developed, resulting in whole plant death. In seedling plants, typical damping-off symptoms were observed. To identify the pathogen, the roots of diseased plants were sampled, surface sterilized, and cultured on potato dextrose agar (PDA) media. From the culture, 4 different fungal isolates were recovered and purely cultured. Each fungal isolate showed distinct growth shapes and color development on malt extract agar, oatmeal agar, sabouraud dextrose agar, and PDA media. Microscopic observation and molecular identification using ribosomal DNA internal transcribed spacer sequencing identified them as 3 Fusarium spp. and 1 Thielaviopsis paradoxa. Additional sequencing of elongation factor 1-alpha and β-tubulin regions of 3 Fusarium spp. revealed that 2 of them are Fusarium solani, and the other one is Fusarium proliferatum. To examine which isolate can act as a causal agent of wilt disease of hemp, each isolate was tested for their pathogenicity. In the pathogenicity test, F. solani AMCF1 and AMCF2, and F. proliferatum AMCF3, but not T. paradoxa AMCF4, were able to cause wilting disease in hemp seedlings. Therefore, we report that F. solani AMCF1 and AMCF2, and F. proliferatum AMCF3 as causal agents of Fusarium wilt of hemp plants. To our knowledge, this is the first report of the wilt disease of C. sativa L. caused by Fusarium spp. in Korea.

New Anti-Hypoxic Metabolites from Co-Culture of Marine-Derived Fungi Aspergillus carneus KMM 4638 and Amphichorda sp. KMM 4639.

The KMM 4639 strain was identified as Amphichorda sp. based on two molecular genetic markers: ITS and β-tubulin regions. Chemical investigation of co-culture marine-derived fungi Amphichorda sp. KMM 4639 and Aspergillus carneus KMM 4638 led to the identification of five new quinazolinone alkaloids felicarnezolines A-E (1-5), a new highly oxygenated chromene derivative oxirapentyn M (6) and five previously reported related compounds. Their structures were established using spectroscopic methods and by comparison with related known compounds. The isolated compounds showed low cytotoxicity against human prostate and breast cancer cells but felicarnezoline B (2) protected rat cardiomyocytes H9c2 and human neuroblastoma SH-SY5Y cells against CoCl2-induced damage.

First Report of Diaporthe eres and D. unshiuensis causing Leaf Spots on Sapindus mukorossi in China.

Sapindus mukorossi Gaertn., commonly known as soapberry, is widely cultivated as a landscaping tree in Southern China. In June 2019, a foliar disease with an incidence of ∼60% occurred on trees was observed in the soapberry germplasm repository, Jianning, Sanming, Fujian, China. The symptoms initially appeared as irregular small yellow spots, while the center of the lesions became dark brown with time. Fragments (size 3 to 4 mm2) taken from lesion margins were sterilized and cultured based on Wang et al. Two isolates (FJ1 and FJ21) were obtained with the following morphological characteristics on PDA, (1) FJ1: Conidiogenous cells were 9.7 to 25.0 × 1.5 to 2.2 μm (n=20). Alpha conidia were 6.1 to 8.3 × 2.2 to 3.0 μm (n=30), aseptate, hyaline, smooth, ellipsoidal. Beta conidia were 28.3 to 38.2 × 1.3 to 1.7 μm (n=30), hyaline, smooth, curved to hooked. Conidial drops were milky colored; (2) FJ21: Pycnidia were dark brown, 280 to 843 μm (n=30) in diam., globose, or irregular on alfalfa stems. Conidiophores were hyaline, cylindrical, smooth, and slightly tapered to the apex, 17.4 to 35.4 × 1.5 to 2.6 μm (n=20). Conidiogenous cells were 14.7 to 29.7 × 1.4 to 2.6 μm (n=20). Alpha conidia were 5.6 to 7.1 × 2.4 to 3.4 μm (n= 30), hyaline, smooth, ellipsoidal, or clavate, aseptate, biguttulate. Beta conidia not observed. Conidial drops were yellow. The morphological characteristics of FJ1 and FJ21 were similar to those of Diaporthe spp.. DNA of two isolates was extracted, and the internal transcribed spacer region (ITS) and partial sequences of translation elongation factor 1-alpha (TEF1-α), calmodulin (CAL), β-tubulin (TUB), and histone H3 (HIS) genes were amplified with primers ITS1/ITS4, EF1-728F/EF1-986R, CAL228F/CAL737R, βt2a/βt2b, and CYLH3F/H3-1b, respectively. The sequences were deposited in GenBank (accession nos. MW585608 and MW768905 to MW768908 for FJ1; MT755625 and MT776728 to MT776731 for FJ21). The BLASTn results showed that the ITS, TEF1-α, TUB, HIS, and CAL sequences of FJ1 were 100, 99, 98, 98, and 99% identical to those of D. eres (NR144923, KJ210550, KJ420799, KJ420850, and KJ434999, respectively). For FJ21, BLASTing with the same loci showed 100, 100, 100, 99, and 100% similarity with those of D. unshiuensis (MH121530, MH121572, MH121607 MH121488, and MH121448, respectively). Phylogenetic analyses with the concatenated sequences placed FJ1 and FJ21 in the clades of D. eres and D. unshiuensis, respectively. Pathogenicity tests were performed by wounding leaves of 2-year-old soapberry seedlings with a sterile needle. The leaves were inoculated with D. eres and D. unshiuensis isolates, respectively, with 10 µl of conidial suspensions (106 conidia/ml). Three plants were used for each treatment, and the leaves of each plant were inoculated. The control was treated with 10 µl of sterile water. The plants were kept in a greenhouse (RH > 80%, 25 ± 2°C). In 5 days, all inoculated leaves showed lesions similar to the field symptoms. Controls were asymptomatic. Diaporthe eres and D. unshiuensis were reisolated from the diseased leaves. No fungus was isolated from the control. Previously, D. biconispora and D. sapindicola were reported as the causal agents of soapberry, but this is the first report of D. eres and D. unshiuensis causing leaf spots on S. mukorossi in China. These data will help develop effective strategies for managing this disease.

First Report of Fusarium falciforme Causing Leaf Blight in Acacia mangium in China.

Acacia mangium Willd. is an important economic tree species with diverse uses and high ecological value, used in agriculture, restoration of degraded lands, for ornamental purposes, and commercial forestry (Leroy et al. 2009). This tree is widely cultivated in tropical and subtropical regions of China, such as Hainan, Guangdong, and Guangxi Provinces. In July 2021, a leaf blight of A. mangium saplings was observed with widespread distribution in the cultivation base of Hainan University (20° 3' 33″ N, 110°18' 56″ E) in Hainan Province, China. The initial symptoms were brown-to-black, irregular-shaped lesions on the leaf margin or tip. The entire leaf wilted and turned brown, leading to death and defoliation in the later periods of the disease (Fig. 1A-C). Of the 50 trees surveyed, the incidence rate was 68%. Small pieces of diseased tissue were collected from six different infected trees. The tissue surface was disinfested with 75% ethanol for 30 s, and 1% mercury chloride for 1 min, then rinsed three times with sterile distilled water. Infected tissues were placed onto potato dextrose agar (PDA) containing 0.01% streptomycin and incubated at 25°C for 7 days. Seventeen isolates were obtained by single-spore isolation on fresh PDA plates, and 15 isolates were identified morphologically as Fusarium spp. (Leslie et al. 2006). The colony surface was white, and the reverse side yellowish (Fig. 1D-E). Microconidia were oval or kidney-shaped, 6.2 to 9.6 × 2.5 to 6.3 μm (n=60). Macroconidia were sickle-shaped, with three to five septa, 19.5 to 41.2 × 3.7 to 6.8 μm (n=60) (Fig. 1F). For molecular identification, genomic DNA of three isolates (HNKFS01, HNKFS02, and HNKFS03) was extracted using E.Z.N.A.® HP Plant DNA kit (Omega Bio-Tek) and the internal transcribed spacer of rDNA (ITS), translation elongation factor 1-α (EF1-α), RNA polymerase II beta subunit (RPB2), and β-tubulin (TUB) regions were amplified and sequenced using primers ITS1F/ITS4, EF-1/EF-2, RPB2-5F2/11aR, and T1/T2, respectively (O'Donnell et al. 2010; O'Donnell and Cigelnik 1997; White et al. 1990). Sequences were deposited in GenBank for ITS (OM289152 to OM289154), EF1-α (OM289155 to OM289157), RPB2 (ON193365 to ON193367), and TUB (ON193314 to ON193316). Using BLASTn, ITS sequences matched 98.88% (532/538 bp) to F. falciforme (MT114705), EF1-α sequences matched 99.58% (703/706 bp; MK752502), RPB2 sequences matched 98.31% (988/1005 bp; MW691193), and TUB gene sequences matched 99.10% (332/335 bp; OK087483). Polyphasic identification with ITS, EF1-α, RPB2, and TUB sequences revealed a 98.51% match with F. falciforme (NRRL 32729) sequences in Fusarium-ID databases. Pathogenicity of 15 isolates was determined by using healthy one-year-old A. mangium seedlings. Three leaves from each seedling were selected to test. Mycelial plugs of each isolate were inoculated on one leaf (three plugs per leaf). An agar plug without the fungus was placed on a leaf as a control. The pathogenicity tests were repeated twice. After 7 days of incubation in a greenhouse, all leaves inoculated with the pathogen showed black lesions with white flocculent hyphae (Fig. 1H), while control leaves were asymptomatic (Fig. 1G). Typical colonies of F. falciforme were isolated from all inoculated leaves, and identified by morphology and ITS, EF1-α, RPB2, and TUB sequence analysis, fulfilling Koch´s postulates. To our knowledge, this is the first report of F. falciforme causing leaf blight in A. mangium in China and the world. This disease can lead to death of A. mangium, which seriously restricts commercialization, requiring that management strategies be adopted.

First Report of Colletotrichum truncatum Causing Anthracnose of Green Foxtail (Setaria viridis) in China.

Green foxtail (Setaria viridis (L.) Beauv.), belonging to the family Gramineae, is a monocotyledonous plant that is distributed in tropical and subtropical regions of the world. S. viridis is one of the most abundant weeds in corn, soybean, rice and other major crops in China, which competes with crops for light, moisture and nutrients, leading to yield losses. In September 2021, an unknown leaf spot disease was observed on the leaves of S. viridis in many greenhouses of Xinkou town, Xiqing district, Tianjin, China (116.95729, 39.09088), under cloudy and high humid conditions after a week of rain. Over 60% of the weeds were observed with leaf spots in 28 greenhouses of XinKou town. The characteristics of the disease were observed and investigated. Initial symptoms were brown spots of 1 to 5 mms, longitudinal elliptic, round, or spindle-shaped lesions on leaves of S. viridis. These spots continued to spread shortly after the onset of the symptoms. At the late-stage disease, the spots' edges were dark brown and irregular. Eventually, the center of the spots turned grayish-white and became thinner and drier until fracture. To investigate the disease, symptomatic weed leaves were separated and small patches with infected spots were cut out. Diseased tissues (3×3 mm) were disinfected with 75% alcohol for 30s  35s, rinsed three times with sterile distilled water and then placed on potato dextrose agar (PDA) at 25°C with a 12-h photoperiod for 7 days in incubators (RXZ-280C, Ningbo, China). With the pathogen growing on the PDA, three mycelia with uniform morphology were observed, which were named SVCT-01, SVCT-02, and SVCT-03, respectively. These mycelia were transferred and cultured for daily observation. The color of these mycelia on PDA appeared gray at first, which eventually turned to grayish black with numerous black microsclerotia, setae, and a few aerial mycelia after 7 days. The setae were 75 to 120 ×3.5 to 5 μm, with elliptic to claviform appressoria. Conidia were hyaline, falcate, unicellular, 16 to 25 × 2.6 to 3.8 μm (n=50). All characteristics of isolates were consistent with the description of Colletotrichum truncatum (Sutton, 1992). Pathogenicity testing was conducted on 3-leaves-stage S. viridis seedlings. Conidial suspension (106 conidia/mL) of isolates were sprayed on 20 S. viridis seedlings with the suspension of each isolate was sprayed on 10 seedlings. Ten seedlings sprayed with sterilized distilled water were used as the control. Three replicates were performed on each treatment. The treatment plants were maintained in the incubator (25°C, relative humidity > 80%, 12-h photoperiod). Typical leaf spot symptoms were observed on inoculated leaves after 7 days, the control leaves remained symptomless. The fungus reisolated from the lesions of diseased leaves were morphological and molecularly identical to the inoculated isolates. The results echo with Koch's postulates，suggesting that the obtained isolates SVCT-01, SVCT-02 and SVCT-03 are potential pathogen in Setaria viridis. To confirm the species' identity, total genomic DNA of isolates were extracted using a Fungal DNA Kit (GBCBIO, Guangzhou, China). Sequences of internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), and β-tubulin (TUB2) regions were identified via PCR (Guerber et al, 2003; Weir et al, 2012). The sequences of SVCT-01, SVCT-02 and SVCT-03 showed more than 99% homology with Colletotrichum truncatum strains CBS:151.35 (GenBank Accession No. GU227862, GU228254, GU227960, GU228156) (Damm, 2009). The sequences of SVCT-01 were deposited in GenBank as a representative isolate under the accession numbers OL629177, OL627527, OM040388, OM040389. Maximum likelihood trees based on concatenated sequences of the four genes were constructed using MEGA7.0. The results showed that the strains isolated from Setaria viridis were closely related to Colletotrichum truncatum with 100% bootstrap support. According to morphological, pathological characteristics, and multilocus phylogenetic analysis, the isolated strains (SVCT-01, SVCT-02 and SVCT-03) from S. viridis were identified as Colletotrichum truncatum (Weir et al, 2012). Colletotrichum sp. is a significant plant pathogen that was previously reported causing anthracnose on Setaria sp. Up to now, it has been reported that C. graminicola has infected nine species of Setaria sp. Such as Setaria glauca in New Zealand (Pennycook, 1989) and Setaria pumila in Zimbabwe (Lenne, 1990). In 1979s, C.graminicola was obtained from Setaria lutescens in China (Tai, 1979). To our knowledge, this is a new host record for Colletotrichum truncatum causing anthracnose on S. viridis in China. Colletotrichum truncatum spread rapidly and caused serious disease to Setaria viridis. We hope to discovery a biocontrol method against weed on non-host cultivated plants through the production of secondary metabolites by C. truncatum.