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.

Unveiling Cryptic Species Diversity and Genetic Variation of Lasiodiplodia (Botryosphaeriaceae, Botryosphaeriales) Infecting Fruit Crops in Taiwan.

The genus Lasiodiplodia, a member of the family Botryosphaeriaceae, is an important fungal disease genus in agriculture. However, the Lasiodiplodia species survey and genetic diversity in Taiwan remain unclear. This study aimed to investigate the Lasiodiplodia species associated with various fruit species to explore the cryptic Lasiodiplodia species diversity, validate species delimitation, and unveil cryptic genetic diversity. Overall, six Lasiodiplodia species were identified, with several new records of infection identified. Additionally, phylogenetic analyses indicated that the relations of all isolates of L. theobromae might be paraphyletic. They were grouped with L. brasiliense based on Automatic Barcode Gap Discovery (ABGD), Automatic Partitioning (ASAP) and structure-based clustering analyses. These analyses did not provide conclusive evidence for L. brasiliensis as a stable species. It may be necessary to gather more information to clarify the species delineation. The multiple new records of Lasiodiplodia species with high genetic diversity and differentiation revealed that the diversity of Lasiodiplodia in Taiwan was underestimated in the past. We found that L. theobromae has the highest number of haplotypes but the lowest number of haplotype and nucleotide diversities, indicating a recent population expansion. This was supported by the significant negative Tajima's D and Fu and Li's D* tests. The high genetic diversity, low gene flow, and host-associated differentiation of Lasiodiplodia species indicate that they might harbour powerful evolutionary potential in Taiwan. This study provided critical insights into genetic variation, host-associated differentiation, and demography of Lasiodiplodia species, which would be helpful for disease management of related pathogens.

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.

Identification of Cyprodinil + Fludioxonil to Manage Soybean Red Crown Rot Using the Microplate-Based High-Throughput Screening and Pot Assay.

Red crown rot (RCR), caused by the soilborne fungus Calonectria ilicicola, is an emerging soybean disease in Taiwan, and fungicide screening is desired to identify effective management for C. ilicicola. This study screened 11 fungicides, including azoxystrobin, boscalid, cyprodinil, cyprodinil + fludioxonil, difenoconazole, fluopyram, flutolanil, mancozeb, prochloraz, pyraclostrobin, and tebuconazole, for their inhibitory effects on the mycelial growth of 10 C. ilicicola field isolates. Subsequently, a microplate-based high-throughput screening (MHTS) method was established to measure the fungicide sensitivity in a population composed of 80 C. ilicicola isolates to three effective fungicides, cyprodinil + fludioxonil, fluopyram, and tebuconazole. The MHTS was optimized for multiple factors, including the optical scanning pattern, absorption wavelength, conidial concentration, and measurement timing based on the quality controls of Z' factor and the log-phase growth curve. The population mean EC50 estimated by MHTS were 0.14, 2.34, and 2.46 ppm to cyprodinil + fludioxonil, fluopyram, and tebuconazole, respectively. In addition to the in vitro assessment, fungicide efficacy was evaluated by coating cyprodinil + fludioxonil, fluopyram, or tebuconazole on soybean seeds in the pot assay. The results showed that cyprodinil + fludioxonil significantly reduced both postemergence damping-off and disease severity, while fluopyram and tebuconazole reduced only the postemergence damping-off but not disease severity. Based on the MHTS and the pot assay results, this study demonstrated cyprodinil + fludioxonil to be a potential fungicide to manage soybean RCR.

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 Soybean Seedling Disease caused by Rhizoctonia solani AG-7 in Taiwan.

From August to November 2020, reduced emergence and damping-off of soybean seedlings were observed in two fields (Benzhou and Wandan) in Taiwan. Disease incidence was approximately 40% in Benzhou by field scouting. The roots of damping-off seedlings were brown. Affected seedlings could be easily pulled out from the soil and the lesions on the roots/stem were generally dry and sunken. These symptoms suggested the possibility of Rhizoctonia infection. Soil surrounding symptomatic seedlings were collected to bait the potential pathogen and symptomatic plants were used for pathogen isolation. The diseased tissues were washed with tap water and surface-disinfected with 1% bleach before placing on the Dexon selection medium at 26°C for 2 days (Ko and Hora 1971). Hyphae were transferred to potato dextrose agar (PDA), and a brown colony with brown and irregular-shaped sclerotia grew from 90 out of 99 isolates. The hyphae exhibited typical characteristics of Rhizoctonia solani, including a constriction and a septum near the end of branching hyphae (Ajayi-Oyetunde and Bradely, 2018). Two isolates from Benzhou and two isolates from Wandan were tested for their pathogenicity, and eight surface-disinfected seeds were distributed evenly on the water agar plates covered by 2-day-old mycelia at 25°C in dark for 7 days. All isolates caused cotyledon rot and reduced germination. To verify their pathogenicity in pots, double-sterilized sorghum seeds were inoculated with two strains and incubated at 25°C for 2 weeks to be used as fungal inoculum (Ajayi-Oyetunde and Bradely, 2017). A layer of 15 ml of fungal inoculum was placed 5 cm beneath the soil surface in pots. Four soybean seeds were planted approximately 3 cm above the inoculum in each pot. After two weeks, reddish lesions on the hypocotyls or taproots of all seedlings in the inoculated pots were observed, while seedlings in the control pots inoculated with sterile sorghum seeds remained healthy. The pathogen was re-isolated from lesions and had identical morphology to the original isolates. To characterize the fungal identity, the internal transcribed spacer (ITS) was sequenced using the primers ITS1/ITS4 (Sharon et al., 2006). Using BLASTN in the NCBI database, the sequence (GenBank no. MW410857 and MW410858) showed 100% (639/639 bp) similarity to KF907734 and 99.83% (635/636 bp) similarity to AF354099, both belong to R. solani anastomosis group 7 (AG-7) (Hua et al. 2014; Gonzalez et al. 2001). Phylogenetic analysis comparing sequences with different AGs (Ajayi-Oyetunde and Bradely, 2017) grouped our isolates within the AG-7 clade with a 100% bootstrap confidence. In the anastomosis test, an incompatible zonation and unequal mycelial growth rates were observed when AG-7 isolates were paired with an AG-1 IA isolate. On the other hand, the compatible tuft reaction was observed when two AG-7 isolates were paired, and the compatible merge reaction was observed in the self-pairing tests (Macnish et al. 1997). Accordingly, the molecular and morphological characterizations confirmed the causal pathogen as R. solani AG-7. R. solani AG-7 was first reported on radishes in Japan (Homma et al., 1983), first found on carnation in Taiwan (Lo et al., 1990), and in field soils of various crops but not soybean (Chuang, 1997). It was suggested that Rhizoctonia diseases of soybean may be present in Taiwan, but molecular confirmation was lacking (Anonymus, 1979). As R. solani AG-7 causes diseases of soybean in the US and Japan (Baird et al., 1996), the importance of AG-7 as an endemic pathogen of soybean in Taiwan should be recognized and its prevalence determined as a first step to managing this disease.

The Genetic Structure, Virulence, and Fungicide Sensitivity of Fusarium fujikuroi in Taiwan.

The rice disease bakanae, caused by Fusarium fujikuroi Nirenberg, has been present in Taiwan for over a century. To better understand the genetic diversity and structure of F. fujikuroi, a set of 16 polymorphic simple sequence repeat (SSR) markers were newly developed and used to analyze 637 F. fujikuroi isolates collected in 14 cities or counties around Taiwan from 1996 to 2013. On the basis of Bayesian clustering, the isolates were classified into four highly differentiated clusters: cluster B likely derived from the more widespread and genetically diversified clusters A or C, and cluster D was restricted to four cities or counties and may have been introduced from unknown sources genetically distinct from clusters A, B, and C. The coexistence of both mating types (MAT1-1:MAT1-2 = 1:1.88) and the highly diversified vegetative compatibility groups (VCG) (16 VCG among the 21 assessed isolates) suggest the likelihood of sexual reproduction in the field. However, the biased mating type ratios and linkage disequilibrium in the population suggest nonrandom mating between individuals. A significant pattern of isolation by distance was also detected, which implies a geographical restricted gene flow and low dissemination ability of F. fujikuroi. Evaluation of 24 representative isolates on eight rice varieties revealed differential levels of virulence, however no clear pattern of specific variety x isolate interaction was observed. Investigations of the differences in virulence and fungicide sensitivity between 8 early isolates (1998 and 2002) and 52 recent isolates (2012) indicate the evolution of increased resistance to the fungicide prochloraz in F. fujikuroi in Taiwan.

Mechanisms relevant to the enhanced virulence of a dihydroxynaphthalene-melanin metabolically engineered entomopathogen.

The entomopathogenic fungus Metarhizium anisopliae MA05-169 is a transformant strain that has been metabolically engineered to express dihydroxynaphthalene-melanin biosynthesis genes. In contrast to the wild type strain, the transformant displays a greater resistance to environmental stress and a higher virulence toward target insect host. However, the underlying mechanisms for these characteristics remain unclear; hence experiments were initiated to explore the possible mechanism(s) through physiological and molecular approaches. Although both transformant and wild type strains could infect and share the same insect host range, the former germinated faster and produced more appressoria than the latter, both in vivo and in vitro. The transformant showed a significantly shorter median lethal time (LT50) when infecting the diamondback moth (Plutella xylostella) and the striped flea beetle (Phyllotreta striolata), than the wild type. Additionally, the transformant was more tolerant to reactive oxygen species (ROS), produced 40-fold more orthosporin and notably overexpressed the transcripts of the pathogenicity-relevant hydrolytic enzymes (chitinase, protease, and phospholipase) genes in vivo. In contrast, appressorium turgor pressure and destruxin A content were slightly decreased compared to the wild type. The transformant's high anti-stress tolerance, its high virulence against five important insect pests (cowpea aphid Aphis craccivora, diamondback moth Pl. xylostella, striped flea beetle Ph. striolata, and silverleaf whitefly Bemisia argentifolii) and its capacity to colonize the root system are key properties for its potential bio-control field application.