Molecular mechanism for endo-type action of glycoside hydrolase family 55 endo-ß-1,3-glucanase on ß1-3/1-6-glucan.

The glycoside hydrolase family 55 (GH55) includes inverting exo-ß-1,3-glucosidases and endo-ß-1,3-glucanases, acting on laminarin, which is a ß1-3/1-6-glucan consisting of a ß1-3/1-6-linked main chain and ß1-6-linked branches. Despite their different modes of action toward laminarin, endo-ß-1,3-glucanases share with exo-ß-1,3-glucosidases conserved residues that form the dead-end structure of subsite -1. Here, we investigated the mechanism of endo-type action on laminarin by GH55 endo-ß-1,3-glucanase MnLam55A, identified from Microdochium nivale. MnLam55A, like other endo-ß-1,3-glucanases, degraded internal ß-d-glucosidic linkages of laminarin, producing more reducing sugars than the sum of d-glucose and gentiooligosaccharides detected. ß1-3-Glucans lacking ß1-6-linkages in the main chain were not hydrolyzed. NMR analysis of the initial degradation of laminarin revealed that MnLam55A preferentially cleaved the nonreducing terminal ß1-3-linkage of the laminarioligosaccharide moiety at the reducing end side of the main chain ß1-6-linkage. MnLam55A liberates d-glucose from laminaritriose and longer laminarioligosaccharides, but kcat/Km values to laminarioligosaccharides (≤4.21 s-1 mM-1) were much lower than to laminarin (5920 s-1 mM-1). These results indicate that ß-glucan binding to the minus subsites of MnLam55A, including exclusive binding of the gentiobiosyl moiety to subsites -1 and -2, is required for high hydrolytic activity. A crystal structure of MnLam55A, determined at 2.4 Å resolution, showed that MnLam55A adopts an overall structure and catalytic site similar to those of exo-ß-1,3-glucosidases. However, MnLam55A possesses an extended substrate-binding cleft that is expected to form the minus subsites. Sequence comparison suggested that other endo-type enzymes share the extended cleft. The specific hydrolysis of internal linkages in laminarin is presumably common to GH55 endo-ß-1,3-glucanases.

Identification and genetic analysis of a pervasive 'needle-eye' sperm phenotype in Drosophila sterile hybrid males.

Interspecies hybrid sterility has been extensively studied, especially in the genus Drosophila. Hybrid sterility is more often found in the heterogametic (XY or ZW) sex, a trend called Haldane's rule. Although this phenomenon is pervasive, identification of a common genetic mechanism remains elusive, with modest support found for a range of potential theories. Here, we identify a single precise morphological phenotype, which we call 'needle-eye sperm', that is associated with hybrid sterility in three separate species pairs that span the Drosophila genus. The nature of the phenotype indicates a common point of meiotic failure in sterile hybrid males. We used 10 generations of backcross selection paired with whole-genome pooled sequencing to genetically map the regions underlying the needle-eye (NE) sperm phenotype. Surprisingly, the sterility phenotype was present in ~50% of males even after 10 generations of backcrossing, and only a single region of the X chromosome was associated with sterility in one direction of backcross. Owing to the common phenotype among sterile male hybrids, and the strong effect of individual loci, further exploration of these findings may identify a universal mechanism for the evolution of hybrid sterility.

Sufficient coumarin accumulation improves apple resistance to Cytospora mali under high-potassium status.

Cytospora canker, caused by Cytospora mali, is the most destructive disease in production of apples (Malus domestica). Adding potassium (K) to apple trees can effectively control this disease. However, the underlying mechanisms of apple resistance to C. mali under high-K (HK) status remain unknown. Here, we found that HK (9.30 g/kg) apple tissues exhibited high disease resistance. The resistance was impeded when blocking K channels, leading to susceptibility even under HK conditions. We detected a suite of resistance events in HK apple tissues, including upregulation of resistance genes, callose deposition, and formation of ligno-suberized tissues. Further multiomics revealed that the phenylpropanoid pathway was reprogrammed by increasing K content from low-K (LK, 4.30 g/kg) status, leading to increases of 18 antifungal chemicals. Among them, the physiological concentration of coumarin (1,2-benzopyrone) became sufficient to inhibit C. mali growth in HK tissues, and exogenous application could improve the C. mali resistance of LK apple branches. Transgenic apple calli overexpressing beta-glucosidase 40 (MdBGLU40), which encodes the enzyme for coumarin synthesis, contained higher levels of coumarin and exhibited high resistance to C. mali even under LK conditions. Conversely, the suppression of MdBGLU40 through RNAi reduced coumarin content and resistance in HK apple calli, supporting the importance of coumarin accumulation in vivo for apple resistance. Moreover, we found that the upregulation of transcription factor MdMYB1r1 directly activated MdBGLU40 and the binding affinity of MdMYB1r1 to the MdBGLU40 promoter increased in HK apple tissue, leading to high levels of coumarin and resistance in HK apple. Overall, we found that the accumulation of defensive metabolites strengthened resistance in apple when raising K from insufficient to optimal status, and these results highlight the optimization of K content in fertilization practices as a disease management strategy.

Identifying sesterterpenoids via feature-based molecular networking and small-scale fermentation.

Terpenoids are known for their diverse structures and broad bioactivities with significant potential in pharmaceutical applications. However, natural products with low yields are usually ignored in traditional chemical analysis. Feature-based molecular networking (FBMN) was developed recently to cluster compounds with similar skeletons, which can highlight trace amounts of unknown compounds. Fusoxypene A is a sesterterpene synthesized by Fusarium oxysporum fusoxypene synthase (FoFS) with a unique 5/6/7/3/5 ring system. In this study, the FoFS-containing biosynthetic gene cluster was identified from F. oxysporum FO14005, and an efficient FBMN-based strategy was established to characterize four new sesterterpenoids, fusoxyordienoid A-D (1-4), based on a small-scale fermentation strategy. A cytochrome P450 monooxygenase, FusB, was found to be involved in the functionalization of fusoxypene A at C-17 and C-24 and responsible for the hydroxylation of fusoxyordienoid A at C-1 and C-8. This study highlights the potential of FBMN as a powerful tool for the discovery and characterization of natural compounds with low abundance. KEY POINTS: Combined small-scale fermentation and FBMN for rapid discovery of fusoxyordienoids Characterization of four new fusoxyordienoids with 5/6/7/3/5 ring system Biosynthetic pathway elucidation via tandem expression and substrate feeding.

Identification and Characterization of Colletotrichum Species Associated with Anthracnose Disease of Plum.

Plum (Prunus salicina Lindl.) is commercially cultivated worldwide for the high levels of nutrients in the fruit. In recent years, anthracnose has been severe in some plum planting areas in China, resulting in a large number of necrotic leaves, blight, and premature leaf fall. In this study, anthracnose samples of plum leaves were collected from Hezhou, Guilin, and Lipu in Guangxi Province and Meishan, Abe Tibetan, and Qiang Autonomous Prefecture of Sichuan Province. Characteristics of mycelia on potato dextrose agar, morphology of appressoria and conidia, and analysis of sequences of several marker regions (internal transcribed spacer [ITS] region, glyceraldehyde-3-phosphate dehydrogenase [GAPDH], chitin synthase [CHS-1], histone H3 [HIS3], actin [ACT], ß-tubulin [TUB2], and the intergenic region between apn2 and MAT1-2-1 [ApMat]). The resulting 101 Colletotrichum isolates obtained were identified as eight species: C. fructicola (50.5%), C. siamense (24.8%), C. karsti (8.9%), C. plurivorum (7.9%), C. aeschynomenes (3.9%), C. gloeosporioides (2%), C. celtidis (1%), and C. phyllanthi (1%). Representatives of all eight Colletotrichum species were found to cause disease on wounded leaves of plum seedlings in pathogenicity assays. As far as we are aware, this is the first report of anthracnose of plum caused by C. celtidis and C. phyllanthi in China.

Fruit Anthracnose on Cavendish bananas Caused by Colletotrichum fructicola in Guangxi, China.

Cavendish banana (Musa spp. AAA group) is one of the main fruit crops worldwide. It is widely planted in Guangdong, Hainan, Guangxi, Fujian and Yunnan provinces in southern China. In November 2020, banana fruits with anthracnose symptoms were collected from Dayu Town (N 23.17°, E 109.80°), Guigang City, and Chengjun Town (N 22.60°, E 110.00°), Yulin City, Guangxi Province, China, where the disease was found on about 70% of the banana plants, and on individual fruit, up to 10% of the surface was covered with symptoms. The symptoms initially began with rust-colored spots on the surface of the immature fruit, which gradually became sunken and cracked as the disease progressed. Small tissues (5×5 mm) from the pericarp at the junction of disease and health were surface-disinfected in 75% ethanol for 10 s, 2% sodium hypochlorite (NaClO) for 1 min, and washed three times in sterile water. Tissue pieces were placed on potato dextrose ager (PDA) and incubated at 25°C. Fifty-nine morphologically similar colonies were obtained after 5 days of incubation, with 100% isolation frequency. Of 59 isolates, GG1-3 isolated from Guigang City and YL4-2 isolated from Yulin City were selected as representative strains for intensive study. Mycelia were off-white for both isolates and conidia obtained from PDA were cylindrical, unicellular, hyaline and obtuse ends, with sizes of 11.5 ± 1.8×3.9 ± 0.8 µm (n=60) and 11.5 ± 1.6×4.1 ± 0.6 µm (n=60) for GG1-3 and YL4-2, respectively (Prihastuti et al. 2009). Genomic DNA was extracted from 7-day-old aerial mycelia using a DNAsecure Plant Kit (Tiangen Biotech, China). The internal transcribed spacer (ITS), the intergenic region of apn2 and MAT1-2-1 (ApMAT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified and sequenced (White et al. 1990; Silva et al.2012; Templeton et al. 1992). Sequences were deposited in GenBank (ITS, OR596961 to OR596962; GAPDH, OR661771 to OR661772; APMAT, OR661773 to OR661774) and showed 100% identities with the corresponding type strains sequences of C. fructicola. Phylogenetic tree was constructed with software raxmlGUI v.2.0.0 based on sequences of multiple loci (ITS, GAPDH and ApMAT) and Maximum Likelihood method. Phylogenetic analysis revealed that the two isolates and C. fructicola were clustered in the same clade, with 94% bootstrap support. According to morphology and phylogenetic analysis, the two isolates GG1-3 and YL4-2 were identified as C. fructicola. For pathogenicity tests, healthy fruits were surface sterilized with 75% ethanol followed by a wash with sterilized water. Five adjacent needle punctures in a 5-mm-diameter circle were made with a sterilized needle on healthy fruits, followed by inoculation with 20 µL of conidial suspension (106 spores/ml), and sterilized water was used as controls. All banana fruit were incubated in a humid chamber at 28°C. After 4 days, all inoculated fruits showed visible symptoms and had rust-colored spots on the margins, while control banana fruits remained symptomless. The fungus was isolated from the inoculated fruit and the isolates were found to match the morphological and molecular characteristics of the original isolates, confirming Koch's hypothesis. To our knowledge, this is the first report of fruit anthracnose on Cavendish bananas caused by C. fructicola in China. This study will provide valuable information for prevention and management of anthracnose on banana fruit.

Peduncle Rot of Banana Caused by Fusarium petroliphilum and F. pernambucanum in Guangxi, China.

Banana is one of the main fruit crops worldwide. In October 2020, peduncles with rot were observed on bananas (Musa sp. ABB, Pisang Awak subgroup) at a about 1600 square meter commercial banana plantation in Dayu Town (23.17° N, 109.80° E), Guigang, Guangxi, China. The incidence of the disease was about 40%. The interior of the peduncle initially appeared reddish-brown and gradually turned black, and the peduncle eventually rotted. Two whole diseased bunches diseased samples were collected from banana plantations. Small pieces of tissues from the peduncle at the junction of disease and health were surface-disinfected in 75% ethanol for 10 s, 2% NaClO for 1 min, and rinsed three times in sterile water, then placed on potato dextrose agar (PDA) for incubation at 25°C. Forty-nine fungal isolates with similar morphology were recovered from diseased tissues, with 82% isolation frequency. Six isolates (GG3-1, GG3-2, GG3-3, GG4-1, GG4-2 and GG4-3) were selected for further study. Genomic DNAs of these isolates were extracted from 7-day-old mycelia. The internal transcribed spacer (ITS), translation elongation factor (TEF1), calmodulin (CAM), and partial RNA polymerase second largest subunit (RPB2) of six representative isolates were amplified and sequenced (O'Donnell et al. 2000, 2010; White et al. 1990). Sequences were deposited in GenBank. (accessions PP087392-PP087397 for ITS; PP102792-PP102797 for TEF1; PP102798-PP102803 for CAM; PP102804-P102809 for RPB2). A phylogenetic tree based on concatenated sequences of all markers using the Maximum Likelihood algorithm (Xia et al. 2019; Schroers et al. 2016). Based on phylogenetic analyses, GG3-1, -2 and -3 were identified as F. petroliphilum with 100% bootstrap support, and GG4-1, -2 and -3 were tightly clustered with F. pernambucanum with 95% bootstrap support. The two representative isolates GG3-2 and GG4-2 were selected for morphology and pathogenicity observation. Colonies of GG3-2 were light yellow and flat mycelium. They produced falciform macroconidia of 46.1 ± 5.3 × 2.6 ± 0.4 µm with 3 to 5 septates, and hyaline, ovoid microconidia of 7.6 ± 0.9 × 4.0 ± 0.6 µm with 0 septate (Brown et al. 2022). Mycelia were whitish to yellowish aerial mycelium for GG4-2. Their macroconidia were falcate of 31.6 ± 3.0 × 4.3 ± 0.3 µm with curved apical cells, foot-shaped basal cells, and 3 to 5 septates. The microconidia were fusoid of 8.9 ± 1.0 × 2.7 ± 0.3 µm with 0 to 1 septate (Santos et al. 2019). For pathogenicity tests, the ends of the banana peduncles were cut off. Needle punctures were made on the ethanol-treated peduncle pieces, followed by inoculation with 20 µL of conidial suspension (106 spores/ml) of each of the two isolates with three replications each. Sterilized water was used as a control. Peduncle pieces were placed in a humid box and incubated at 28ºC. After 7 days, reddish-brown to black lesions were observed on all inoculated peduncle pieces, while no symptoms were observed on the control pieces. The fungus was isolated from the inoculated peduncle pieces and found to match the morphological characteristics and marker sequences of the original isolates, confirming Koch's postulates. To our knowledge, this is the first report of peduncle rot on banana caused by F. petroliphilum and F. pernambucanum in China. This study will provide valuable information on causal pathogens of this disease which can contribute to improving prevention and disease management strategies for growers.

Function Switch of a Fungal Sesterterpene Synthase through Molecular Dynamics Simulation Assisted Alteration of an Aromatic Residue Cluster in the Active Pocket of PfNS.

Terpene synthases (TPSs) play pivotal roles in generating diverse terpenoids through complex cyclization pathways. Protein engineering of TPSs offers a crucial approach to expanding terpene diversity. However, significant potential remains untapped due to limited understanding of the structure-function relationships of TPSs. In this investigation, using a joint approach of molecular dynamics simulations-assisted engineering and site-directed mutagenesis, we manipulated the aromatic residue cluster (ARC) of a bifunctional terpene synthase (BFTPS), Pestalotiopsis fici nigtetraene synthase (PfNS). This led to the discovery of previously unreported catalytic functions yielding different cyclization patterns of sesterterpenes. Specifically, a quadruple variant (F89A/Y113F/W193L/T194W) completely altered PfNS's function, converting it from producing the bicyclic sesterterpene nigtetraene to the tricyclic ophiobolin F. Additionally, analysis of catalytic profiles by double, triple, and quadruple variants demonstrated that the ARC functions as a switch, unprecedently redirecting the production of 5/11 bicyclic (Type B) sesterterpenes to 5/15 bicyclic (Type A) ones. Molecular dynamics simulations and theozyme calculations further elucidated that, in addition to cation-π interactions, C-H⋅⋅⋅π interactions also play a key role in the cyclization patterns. This study offers a feasible strategy in protein engineering of TPSs for various industrial applications.

A secreted fungal subtilase interferes with rice immunity via degradation of SUPPRESSOR OF G2 ALLELE OF skp1.

Serine protease subtilase, found widely in both eukaryotes and prokaryotes, participates in various biological processes. However, how fungal subtilase regulates plant immunity is a major concern. Here, we identified a secreted fungal subtilase, UvPr1a, from the rice false smut (RFS) fungus Ustilaginoidea virens. We characterized UvPr1a as a virulence effector localized to the plant cytoplasm that inhibits plant cell death induced by Bax. Heterologous expression of UvPr1a in rice (Oryza sativa) enhanced plant susceptibility to rice pathogens. UvPr1a interacted with the important rice protein SUPPRESSOR OF G2 ALLELE OF skp1 (OsSGT1), a positive regulator of innate immunity against multiple rice pathogens, degrading OsSGT1 in a protease activity-dependent manner. Furthermore, host-induced gene silencing of UvPr1a compromised disease resistance of rice plants. Our work reveals a previously uncharacterized fungal virulence strategy in which a fungal pathogen secretes a subtilase to interfere with rice immunity through degradation of OsSGT1, thereby promoting infection. These genetic resources provide tools for introducing RFS resistance and further our understanding of plant-pathogen interactions.

Complete genome sequence of a novel chrysovirus infecting Aspergillus terreus.

A double-stranded RNA (dsRNA) mycovirus was obtained from Aspergillus terreus strain HJ3-26 and designated "Aspergillus terreus chrysovirus 1" (AtCV1). It consists of four dsRNA segments (dsRNA1-4) with lengths of 3612 bp, 3132 bp, 3153 bp, and 3144 bp, respectively. Sequence analysis showed that dsRNA1 encodes an RNA-dependent RNA polymerase (RdRp), dsRNA2 encodes a capsid protein, and both dsRNA3 and dsRNA4 encode hypothetical proteins. Phylogenetic analysis of the RdRp suggested that AtCV1 is a member of a new species of the genus Alphachrysovirus in the family Chrysoviridae. This is the first chrysovirus obtained from A. terreus.

Identification and characterization of extracellular GH3 ß-glucosidase from the pink snow mold fungus, Microdochium nivale.

Glycoside hydrolase family 3 (GH3) ß-glucosidase exists in many filamentous fungi. In phytopathogenic fungi, it is involved in fungal growth and pathogenicity. Microdochium nivale is a severe phytopathogenic fungus of grasses and cereals and is the causal agent of pink snow mold, but its ß-glucosidase has not been identified. In this study, a GH3 ß-glucosidase of M. nivale (MnBG3A) was identified and characterized. Among various p-nitrophenyl ß-glycosides, MnBG3A showed activity on d-glucoside (pNP-Glc) and slight activity on d-xyloside. In the pNP-Glc hydrolysis, substrate inhibition occurred (Kis = 1.6 m m), and d-glucose caused competitive inhibition (Ki = 0.5 m m). MnBG3A acted on ß-glucobioses with ß1-3, -6, -4, and -2 linkages, in descending order of kcat/Km. In contrast, the regioselectivity for newly formed products was limited to ß1-6 linkage. MnBG3A has similar features to those of ß-glucosidases from Aspergillus spp., but higher sensitivity to inhibitory effects.

Ornithine decarboxylase of the fungal pathogen Colletotrichum higginsianum plays an important role in regulating global metabolic pathways and virulence.

Colletotrichum higginsianum is an important fungal pathogen causing anthracnose disease of cruciferous plants. In this study, we characterized a putative orthologue of yeast SPE1 in C. higginsianum, named ChODC. Deletion mutants of ChODC were defective in hyphal and conidial development. Importantly, deletion of ChODC significantly affected appressorium-mediated penetration in C. higginsianum. However, polyamines partially restore appressorium function and virulence indicating that loss of ChODC caused significantly decreased virulence by the crosstalk between polyamines and other metabolic pathways. Subsequently, transcriptomic and metabolomic analyses demonstrated that ChODC played an important role in metabolism of various carbon and nitrogen compounds including amino acids, carbohydrates and lipids. Along with these clues, we found deletion of ChODC affected glycogen and lipid metabolism, which were important for conidial storage utilization and functional appressorium formation. Loss of ChODC affected the mTOR signalling pathway via modulation of autophagy. Interestingly, cAMP treatment restored functional appressoria to the ΔChODC mutant, and rapamycin treatment also stimulated formation of functional appressoria in the ΔChODC mutant. Overall, ChODC was associated with the polyamine biosynthesis pathway, as a mediator of cAMP and mTOR signalling pathways to regulate appressorium function. Our study provides evidence of a link between ChODC and the cAMP signalling pathway and defines a novel mechanism by which ChODC regulates infection-associated autophagy and plant infection by fungi.

A secreted fungal effector suppresses rice immunity through host histone hypoacetylation.

Histone acetylation is a critical epigenetic modification that regulates plant immunity. Fungal pathogens secrete effectors that modulate host immunity and facilitate infection, but whether fungal pathogens have evolved effectors that directly target plant histone acetylation remains unknown. Here, we identified a secreted protein, UvSec117, from the rice false smut fungus, Ustilaginoidea virens, as a key effector that can target the rice histone deacetylase OsHDA701 and negatively regulates rice broad-spectrum resistance against rice pathogens. UvSec117 disrupts host immunity by recruiting OsHDA701 to the nucleus and enhancing OsHDA701-modulated deacetylation, thereby reducing histone H3K9 acetylation levels in rice plants and interfering with defense gene activation. Host-induced gene silencing of UvSec117 promotes rice resistance to U. virens, thus providing an alternative way for developing rice false smut-resistant plants. This is the first direct evidence demonstrating that a fungal effector targets a histone deacetylase to suppress plant immunity. Our data provided insight into a counter-defense mechanism in a plant pathogen that inactivates host defense responses at the epigenetic level.

Complete genome sequence of a novel chrysovirus infecting Talaromyces neofusisporus.

A double-stranded RNA (dsRNA) mycovirus was isolated from Talaromyces neofusisporus isolate HJ1-6 and named "Talaromyces neofusisporus chrysovirus 1" (TnCV1). It was found to consist of four dsRNA segments (TnCV1-1, TnCV1-2, TnCV1-3, and TnCV1-4) with lengths of 3595 bp, 3063 bp, 3054 bp, and 2876 bp, respectively. Sequence analysis showed that TnCV1-1 contains an open reading frame (ORF) encoding a putative RNA-dependent RNA polymerase (RdRp) of 1136 amino acids (aa), TnCV1-2 contains an ORF encoding a hypothetical protein of 906 aa, TnCV1-3 contains an ORF encoding a putative capsid protein (CP) of 938 aa, and TnCV1-4 contains an ORF encoding a hypothetical protein of 849 aa. The 5' and 3' untranslated regions (UTRs) of TnCV1-1, TnCV1-2, TnCV1-3, and TnCV1-4 showed a high degree of sequence similarity to each other. Phylogenetic analysis based on RdRp sequences suggested that TnCV1 is a new member of the genus Alphachrysovirus in the family Chrysoviridae. This is the first chrysovirus isolated from T. neofusisporus.

Complete genome sequence of a novel victorivirus infecting Aspergillus niger.

Aspergillus niger is an important filamentous phytopathogenic fungus with a broad host range. A novel double-stranded (ds) RNA mycovirus, named Aspergillus niger victorivirus 1 (AnV1), isolated from A. niger strain baiyun3.23-4, was sequenced and analyzed. The AnV1 genome is 5317 nucleotides long with a GC content of 56%. AnV1 contains two open reading frames (ORF1 and 2), overlapping at a tetranucleotide sequence (AUGA). ORF1 encodes a putative capsid protein (CP) of 778 amino acids (aa), while ORF2 potentially encodes a putative RNA-dependent RNA polymerase (RdRp) of 826 aa. Phylogenetic analysis indicated that AnV1 is a new member of the genus Victorivirus in the family Totiviridae. As far as we know, this is the first report of the complete genome sequence of a victorivirus infecting A. niger.

Schultriene and nigtetraene: two sesterterpenes characterized from pathogenetic fungi via genome mining approach.

Fungal bifunctional terpene synthases (BFTSs) have been reported to contribute to the biosynthesis of a variety of di/sesterterpenes via different carbocation transportation pathways. Genome mining of new BFTSs from unique fungal resources will, theoretically, allow for the identification of new terpenes. In this study, we surveyed the distribution of BFTSs in our in-house collection of 430 pathogenetic fungi and preferred two BFTSs (CsSS and NnNS), long distance from previously characterized BFTSs and located in relatively independent branches, based on the established phylogenetic tree. The heterologous expression of the two BFTSs in Aspergillus oryzae and Saccharomyces cerevisiae led to the identification of two new sesterterpenes separately, 5/12/5 tricyclic type-A sesterterpene (schultriene, 1) for CsSS and 5/11 bicyclic type-B sesterterpene (nigtetraene, 2) for NnNS. In addition, to the best of our knowledge, 2 is the first 5/11 bicyclic type-B characterized sesterterpene to date. On the basis of this, the plausible cyclization mechanisms of 1 and 2 were proposed based on density functional theory calculations. These new enzymes and their corresponding terpenes suggest that the chemical spaces produced by BFTSs remain large and also provide important evidences for further protein engineering for new terpenes and for understanding of cyclization mechanism catalyzed by BFTSs. KEY POINTS: • Genome mining of two BFTSs yields two new sesterterpenoids correspondingly. • Identification of the first 5/11 ring system type-B product. • Parse out the rational cyclization mechanism of isolated sesterterpenoids.

First report of leaf spot and stem blight on blueberry (Vaccinium corymbosum cv. Bluerain) caused by Calonectria pseudoreteaudii in China.

Blueberry has high nutritional value and is one of the five healthy fruits. In 2018, leaf spots and stem blights were observed on Vaccinium corymbosum cv. Bluerain in Guangzhou, Guangdong Province, China. Up to 80% of the plants were affected. Initial symptoms of affected leaves were red-brown, irregular, small spots, which gradually coalesced and formed larger irregular necrotic patches. The affected stems showed red-brown and irregular large lesions. Diseased tissues were surface sterilized with 75% alcohol for 15 s, followed by 2.5% NaClO for 30 s, and rinsing three times in sterile distilled water, placed on potato dextrose agar (PDA) and incubated at 25 C. Representative strains, ZHKUCC 21-0021 from diseased leaves and ZHKUCC 21-0073 from diseased stems, were selected for further studies. Colonies grew slowly at 25 C on malt extract agar (MEA) (average 5.68 mm/d), producing white aerial mycelium and red-brown color on the underside after 7 days. Macroconidiophores were hyaline, smooth, consisting of a stipe bearing fertile branches, and a stipe extension terminating in a vesicle. Each terminal branch produced 2-4 phialides, 8-13 × 3-6 µm, reniform or doliiform; Stipe extensions were septate, terminating in a narrowly clavate vesicle, 2-6 µm. Macroconidia were hyaline, straight cylindrical, round at both ends, 83-100 × 7-11 µm (average = 94 × 8 µm; n = 50), with 5 septa. These morphological characteristics were similar to the description of Calonectria pseudoreteaudii (Lombard et al., 2010). The partial calmodulin (cmdA), beta-tubulin (ß-tubulin), and translation elongation factor 1-alpha (tef1-α) genes of the two isolates were respectively amplified using primers CAL-228F/CAL-737R (Carbone et al., 1999), EF1-728F/EF2 and T1/CYLTUB1R (Lombard et al., 2015), and sequences were deposited in GenBank (cmdA: MZ516854 and MZ516855; ß-tubulin: MZ516858 and MZ516859; tef1-α: MZ516856 and MZ516857). BLAST analysis of three gene sequences showed 100% similarity to those of C. pseudoreteaudii. In the maximum likelihood (ML) tree of the concatenated sequences of the three genes, the two isolates from this study were clustered with C. pseudoreteaudii with 100% bootstrap support. Five-mm-diameter hyphal plugs of two representative isolates grown on PDA for five days were used in the pathogenicity test. Leaves were inoculated with ZHKUCC 21-0021, and stems were inoculated with ZHKUCC 21-0073 with five replicates. As controls, sterile PDA plugs were used. All inoculated plants were maintained at 25 C . After 7 days, inoculated leaves and stems developed symptoms similar to field samples, whereas the control plants remained asymptomatic. The pathogen was reisolated from inoculated plants and confirmed to be C. pseudoreteaudii by morphological characteristics. Five Calonectria species (C. canadensis, C. colhounii, C. ilicicola, C. kyotensis and C. pyrochroa), have been reported associated with blueberry (Farr and Rossman, 2022; Fei et al, 2017). Calonectria canadensis and C. ilicicola have been reported to cause stem blight and stem rot in Vaccinium spp. in China (Fei et al, 2017 and 2018). Calonectria colhounii has been reported to cause stem blight in V. angustifolium and V. corymbosum in the United States (Sadowsky et al, 2011). However, this is the first report of C. pseudoreteaudii causing leaf spot and stem blight on Vaccinium spp. worldwide. These results will provide a foundation for future research on prevention and control of this disease.

Draft Genome Sequence of Alternaria longipes Causing Tobacco Brown Spot.

Alternaria is a cosmopolitan fungal genus associated with diverse hosts. Tobacco brown spot caused by Alternaria longipes is one of the most destructive diseases of tobacco. A. longipes can also infect many other plants, some animals and even humans. Here, we report a genome assembly of A. longipes CBS 540.94 using Oxford Nanopore Technologies. A total of 15 contigs were assembled, and the genome size was 37.5 Mb with contig N50 of 4.33 Mb. This genome resource will provide information for further research on comparative genomics of the genus Alternaria and be a valuable resource in investigations of the molecular interactions of pathogen and hosts.

Identification and Observation of Infection Processes of Colletotrichum Species Associated with Pearl Plum Anthracnose in Guangxi, China.

Pearl plum (Prunus salicina Lindl.) is mainly cultivated in Tian'e County in Guangxi Province, southern China. Anthracnose is a devastating disease on pearl plum, causing extensive leaf blight. Diseased leaves were sampled from 21 orchards in Tian'e County. Isolates were first screened for ones resembling Colletotrichum, and 21 representative isolates were selected for sequencing of portions of the ribosomal internal transcribed spacer (ITS), the intergenic region of apn2 and MAT1-2-1 genes (ApMAT), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), calmodulin (CAL), chitin synthase (CHS-1), and ß-tubulin 2 (TUB2). Based on colony, conidial, and appressorial morphology and sequence analyses, the Colletotrichum isolates associated with pearl plum anthracnose were identified as four species: Colletotrichum fructicola (16 isolates), C. gloeosporioides (3 isolates), C. cigarro (1 isolate), and C. siamense (1 isolate). The results of pathogenicity tests showed that isolates of all four species were pathogenic to wounded leaves of pearl plum seedlings. In this study, we microscopically observed the infection processes of isolates of these four species on attached pearl plum leaves. For C. cigarro and C. siamense, the entire infection processes took 120 h; for C. fructicola and C. gloeosporioides, it only took 72 h. This is the first report of C. fructicola and C. cigarro causing anthracnose on pearl plum worldwide, and also the first report of C. siamense causing anthracnose on pearl plum in China.

ATAC-Seq Data for Genome-Wide Profiling of Transcription Factor Binding Sites in the Rice False Smut Fungus Ustilaginoidea virens.

Identification of transcription factor binding sites is one of the most important steps in understanding the function of transcription factors and regulatory networks in organisms. The assay for transposase accessible chromatin sequencing (ATAC-seq) is a simple protocol for detection of open chromatin that could be a powerful tool to advance studies of protein-DNA interactions. Although ATAC-seq has been used in systematic identification of cis-regulatory regions in animal and plant genomes, this method has been rarely applied in fungi. Here, we describe a valuable ATAC-seq resource in the genome of an economically important phytopathogen, the rice false smut fungus Ustilaginoidea virens. The ATAC-seq data of U. virens mycelia collected from potato sucrose broth (PSB) and PSB supplied with rice spikelet extract were both generated. This is the first genome-wide profiling of open chromatin and transcription factor binding sites in U. virens.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.