Ca2+ affects the hyphal differentiation to sclerotia formation of Athelia rolfsii.

RNA-Sequencing (RNA-Seq) and transcriptomic analyses have become powerful tools to study the developmental stages of fungal structures scuh as sclerotia. While RNA-Seq experiments have been set up for many important sclerotia- and microsclerotia-forming fungi, it has not been implemented to study Athelia rolfsii, which is one of the earliest fungi used in literature to uncover the roles of reactive oxygen species (ROS) in stimulating sclerotia formation. This study applied RNA-Seq to profile gene expression in four developmental stages of A. rolfsii sclerotia. Surprisingly, gene ontology and expression patterns suggested that most ROS-scavenging genes were not up-regulated in the stages from hyphal differentiation to the initial sclerotia stage. Using antioxidant and oxidant-amended culture assay, the results suggested none of the ascorbic acid, dithiothreitol (DTT), H2O2, or superoxide dismutase inhibitors [diethyldithiocarbamate (DETC), NaN3, and sodium dodecyl sulfate] affected the sclerotia number. Instead, only glutathione reduced the sclerotia number. Because glutathione has also been suggested to facilitate Ca2+ influx, therefore, glutathione culture assays with the combination of CaCl2, Ca2+-chelator egtazic acid, DETC, and H2O2 were tested on A. rolfsii, as well as two other fungi (Sclerotinia sclerotiorum and Macrophomina phaseolina) for comparison. Although the addition of CaCl2 caused sclerotia or microsclerotia reduction for all three fungi, the CaCl2-ROS interaction was only observed for S. sclerotiorum and M. phaseolina, but not A. rolfsi. Collectively, this study not only pointed out a conserved function of Ca2+ in suppressing fungal sclerotia and microsclerotia formation but also highlighted sclerotia formation of A. rolfsii being only sensitive to Ca2+ and independent of ROS stimuli.IMPORTANCEManagement for plant diseases caused by soil-borne fungal pathogens is challenging because many soil-borne fungal pathogens form sclerotia for long-term survival. Advanced understanding of the molecular and cellular mechanisms of sclerotia formation may provide novel insights to prevent these fungal residues in fields. This study discovered that Ca2+ acts as a negative signal cue to suppress sclerotia and microsclerotia formation in three economically important fungal pathogens. Moreover, the southern blight fungus Athelia rolfsii appears to be only regulated by Ca2+ but not reactive oxygen species. Accordingly, A. rolfsii can be a useful system for studying the detailed mechanism of Ca2+, and the applicability of Ca2+ in reducing sclerotia could be further assessed for disease management.

Heritability and gene functions associated with sclerotia formation of Rhizoctonia solani AG-7 using whole genome sequencing and genome-wide association study.

Sclerotia are specialized fungal structures formed by pigmented and aggregated hyphae, which can survive under unfavourable environmental conditions and serve as the primary inocula for several phytopathogenic fungi including Rhizoctonia solani. Among 154 R. solani anastomosis group 7 (AG-7) isolates collected in fields, the sclerotia-forming capability regarding sclerotia number and sclerotia size varied in the fungal population, but the genetic makeup of these phenotypes remained unclear. As limited studies have focused on the genomics of R. solani AG-7 and the population genetics of sclerotia formation, this study completed the whole genome sequencing and gene prediction of R. solani AG-7 using the Oxford NanoPore and Illumina RNA sequencing. Meanwhile, a high-throughput image-based method was established to quantify the sclerotia-forming capability, and the phenotypic correlation between sclerotia number and sclerotia size was low. A genome-wide association study identified three and five significant SNPs associated with sclerotia number and size in distinct genomic regions, respectively. Of these significant SNPs, two and four showed significant differences in the phenotypic mean separation for sclerotia number and sclerotia size, respectively. Gene ontology enrichment analysis focusing on the linkage disequilibrium blocks of significant SNPs identified more categories related to oxidative stress for sclerotia number, and more categories related to cell development, signalling and metabolism for sclerotia size. These results indicated that different genetic mechanisms may underlie these two phenotypes. Moreover, the heritability of sclerotia number and sclerotia size were estimated for the first time to be 0.92 and 0.31, respectively. This study provides new insights into the heritability and gene functions related to the development of sclerotia number and sclerotia size, which could provide additional knowledge to reduce fungal residues in fields and achieve sustainable disease management.

Superoxide Initiates the Hyphal Differentiation to Microsclerotia Formation of Macrophomina phaseolina.

The infection of Macrophomina phaseolina often results in a grayish appearance with numerous survival structures, microsclerotia, on the plant surface. Past works have studied the development of fungal survival structures, sclerotia and microsclerotia, in the Leotiomycetes and Sordariomycetes. However, M. phaseolina belongs to the Dothideomycetes, and it remains unclear whether the mechanism of microsclerotia formation remains conserved among these phylogenetic clades. This study applied RNA-sequencing (RNA-Seq) to profile gene expressions at four stages of microsclerotia formation, and the results suggested that reactive oxygen species (ROS)-related functions were significantly different between the microsclerotia stages and the hyphal stage. Microsclerotia formation was reduced in the plates amended with antioxidants such as ascorbic acid, dithiothreitol (DTT), and glutathione. Surprisingly, DTT drastically scavenged H2O2, but the microsclerotia amount remained similar to the treatment of ascorbic acid and glutathione that both did not completely eliminate H2O2. This observation suggested the importance of [Formula: see text] over H2O2 in initiating microsclerotia formation. To further validate this hypothesis, the superoxide dismutase 1 (SOD1) inhibitor diethyldithiocarbamate trihydrate (DETC) and H2O2 were tested. The addition of DETC resulted in the accumulation of endogenous [Formula: see text] and more microsclerotia formation, but the treatment of H2O2 did not. The expression of SOD1 genes were also found to be upregulated in the hyphae to the microsclerotia stage, which suggested a higher endogenous [Formula: see text] stress presented in these stages. In summary, this study not only showed that the ROS stimulation remained conserved for initiating microsclerotia formation of M. phaseolina but also highlighted the importance of [Formula: see text] in initiating the hyphal differentiation to microsclerotia formation. IMPORTANCE Reactive oxygen species (ROS) have been proposed as the key stimulus for sclerotia development by studying fungal systems such as Sclerotinia sclerotiorum, and the theory has been adapted for microsclerotia development in Verticillium dahliae and Nomuraea rileyi. While many studies agreed on the association between (micro)sclerotia development and the ROS pathway, which ROS type, superoxide ([Formula: see text]) or hydrogen peroxide (H2O2), plays a major role in initiating hyphal differentiation to the (micro)sclerotia formation remains controversial, and literature supporting either [Formula: see text] or H2O2 can be found. This study confirmed the association between ROS and microsclerotia formation for the charcoal rot fungus Macrophomina phaseolina. Moreover, the accumulation of [Formula: see text] but not H2O2 was found to induce higher density of microsclerotia. By integrating transcriptomic and phenotypic assays, this study presented the first conclusive case for M. phaseolina that [Formula: see text] is the main ROS stimulus in determining the amount of microsclerotia formation.

First Report of Corynespora cassiicola Causing Target Spot on Soybean in Taiwan.

Soybean (Glycine max [L.] Merr.) is an important crop in Taiwan. In October 2020, an unknown leaf spot disease was counted (n = 100) to occur over 70% of soybean cultivar 'Hualien No.1' in the Shoufeng Township of Hualien County, eastern Taiwan. Initial symptoms on leaves as tiny lesions approximately 3 mm in diameter, which later enlarged and developed into round, irregular, and reddish-brown spots with concentric rings surrounded by a yellowish halo. The symptoms appeared on both young and old leaves, but rarely on the stem or pods. The lesions at the margin of healthy and infected tissues were surface-disinfested in 1% NaOCl for 30 seconds, washed twice in sterilized distilled water, dissected and plated on potato dextrose agar (PDA) to isolate the potential pathogen. Colonies on PDA exhibited light to dark brown color at 24°C with 12-hours light after 7-days incubation. The average growth rate was 3 mm per day. Conidia were light brown in color and obclavate to cylindrical in shape. The size of a conidium was measured with an average of 110.8 ± 28.2 µm in length and 15.2 ± 2.8 µm in width, typically with 3 to 18 septa (n = 50). To confirm the pathogenicity of this fungus, conidial suspension (104 conidia/mL) of two isolates, HL_GM-6 and HL_GM-7, were sprayed on the healthy leaves of 4-weeks-old soybean. Plants sprayed with sterile distilled water were used as a control. After inoculation, the plants were covered with plastic bags to maintain a high humidity for 24 hours before moving into a greenhouse with a condition of 20 to 25°C and relative humidity of 75 to 80%. After 7 days of inoculation, foliar symptoms began to appear and which were identical with the field observations. To complete the Koch's postulates, pathogen isolation was attempted and the identical fungus was retrieved from the foliar spots of the inoculated leaves. The foliar symptoms as well as the morphology of the conidiophores and conidia suggested the pathogen to be Corynespora cassiicola (Ellis et al. 1971). Molecular characterization was performed using the sequences of internal transcribed spacer (ITS) region of rDNA, actin (act1), tubulin, and translation elongation factor 1 alpha (tef1) genes after a PCR with ITS1/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn, 1999), BT2a/Bt2b (Udayanga et al. 2012), EF1-728F/EF1-986R (Udayanga et al. 2012), respectively. BLASTN sequence analyses of the ITS, act1, tubulin, and tef1 genomic regions of the isolate HL_GM-7 (GenBanK accessions MW548097 MW961420, MW961419 and MW961421) showed high similarity with the isolates of C. cassiicola including 99.58% with sequence KF810854 (Deon et al. 2014), 99.11% with FJ853005 (Dixon et al. 2009), 99.34% with MH763700 (Duan et al. 2019), and 99.33% with KY112719 (Zhang et al. 2018) respectively. Based on the morphology, pathogenicity, and sequence results, this study becomes the first report of C. cassiicola causing target spot on soybean in Taiwan. C. cassiicola is known to infect a broad host range (Dixon et al. 2009; Lopezet al. 2018), and it has been found to infect tomato, cucumber, papaya, and Salvia miltiorrhiza in Taiwan (Lu et al. 2019; Tsai et al. 2015). Therefore, the emergence of soybean target spot should be aware to avoid potential damage to soybean production in Taiwan.

First Report of Leaf Spot Caused by Diaporthe tulliensis on Boston Ivy (Parthenocissus tricuspidata) in Taiwan.

Starting from the May to August 2020 (average humidity 76.6% and temperature 25.2°C in Taipei), Boston ivy (Parthenocissus tricuspidata) plants on the campus of National Taiwan University (25°01'05.4"N 121°32'36.6"E) exhibited leaf rusts caused by Phakopsora ampelopsidis (Tzean et al., 2019) and leaf spots caused by an unknown pathogen. The leaf spots appeared reddish to brown color and mostly irregular to round shape on the simple and trifoliate leaflets (Supplemental Figure 1A-C). The leaf spots were surface-disinfected with 1% NaOCl for 30 seconds, and the margin of healthy and infected tissues was cut and placed onto water agar, which were incubated at room temperature. Hyphae grown out from leaf spots were sub-cultured on potato dextrose agar (PDA), and the majority of isolates exhibited white colony with black pycnidial conidiomata embedded in PDA. The pycnidial conidiomata of two-week-old has an average diameter of 463±193 µm (n=30) and the sizes of α-conidia were 5.71±0.49 µm in length and 2.42±0.32 µm in width (n=50) similar to the previous records (Crous et al. 2015). The α-conidium was one-celled, hyaline, and ovoid with two droplets (Supplemental Figure 1D-G). This putative pathogen was re-inoculated to confirm its pathogenicity on the leaves of Boston ivy plants. A PDA block with actively growing fungal edge was placed on the tiny needle-wounded leaves of detached branches (Supplemental Figure H-I) and the whole plants in pots (Supplemental Figure 1J-M) in a moist chamber at 28°C in dark. Reddish to brown leaf spots were observed by 2 days post-inoculation (dpi) and the leaf spots expanded by 5 dpi. To complete the Koch's postulates, the pathogen was re-isolated from inoculated leaves and the re-isolated pathogen exhibited identical morphology to the original isolate. The internal transcribed spacer (ITS), translational elongation factor subunit 1-α gene (EF1α), ß-tubulin (BT), and calmodulin (CAL) was amplified using the primers ITS1/ITS4 (Martin and Rygiewicz. 2005), EF1-728F/EF1-986R, Bt2a/Bt2b, and CAL-228F/CAL-737R, respectively (Manawasinghe et al. 2019). Using BLAST in the NCBI database, the ITS (MT974186), EF1α (MT982963), and ß-tubulin (MT982962) sequences showed 98.57% (NR_147574.1, 553 out of 561 bp), 98.04% (KR936133.1, 350 out of 357 bp), and 99.23% (KR936132.1, 518 out of 522 bp) identity to the Diaporthe tulliensis ex-type BRIP 62248a, respectively (Dissanayake et al. 2017). Phylogenetic analysis using concatenated sequences of ITS, EF1α, and ß-tubulin grouped the D. tulliensis isolated from Boston ivy leaf spots with the D. tulliensis ex-type (Supplemental Figure 1N). In summary, the morphological and molecular characterizations supported the causal pathogen of Boston ivy leaf spot as D. tulliensis. While Diaporthe ampelopsidis was reported to infect Parthenocissus quinquefolia and P. tricuspidata (Anonymous, 1960; Wehmeyer, 1933), there is no record for D. tulliensis infecting Boston ivy according to the USDA National Fungus Collections (Farr and Rossman. 2020). Because pathogens of Boston ivy such as P. ampelopsidis may also infect close-related crops like grape (Vitis vinifera L.) and D. tulliensis has been known to infect kiwifruits (Actinidia chinensis) and cocoa (Theobroma cacao) (Bai et al. 2016; Yang et al. 2018), the emergence of D. tulliensis should be aware to avoid potential damage to economic crops.

Whole-Genome Sequence Resource of Calonectria ilicicola, the Casual Pathogen of Soybean Red Crown Rot.

Calonectria ilicicola (anamorph: Cylindrocladium parasiticum) is a soilborne plant-pathogenic fungus with a broad host range, and it can cause red crown rot of soybean and Cylindrocladium black rot of peanut, which has become an emerging threat to crop production worldwide. Limited molecular studies have focused on Calonectria ilicicola and one of the possible difficulties is the lack of genomic resources. This study presents the first high quality and near-completed genome of C. ilicicola, using the Oxford Nanopore GridION sequencing platform. A total of 16 contigs were assembled and the genome of C. ilicicola isolate F018 was estimated to have 11 chromosomes. Currently, the C. ilicicola F018 genome represents the most contiguous assembly, which has the lowest contig number and the highest contig N50 among all Calonectria genome resources. Putative protein-coding sequences and secretory proteins were estimated to be 17,308 and 1,930 in the C. ilicicola F018 genome, respectively; and the prediction was close to other plant-pathogenic fungi, such as Fusarium species, within the Nectriaceae family. The availability of this high-quality genome resource is expected to facilitate research on fungal biology and genetics of C. ilicicola and to support advanced understanding of pathogen virulence and disease management.[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.