Analyzing genetic diversity in luffa and developing a Fusarium wilt-susceptible linked SNP marker through a single plant genome-wide association (sp-GWAS) study.

BACKGROUND: Luffa (Luffa spp.) is an economically important crop of the Cucurbitaceae family, commonly known as sponge gourd or vegetable gourd. It is an annual cross-pollinated crop primarily found in the subtropical and tropical regions of Asia, Australia, Africa, and the Americas. Luffa serves not only as a vegetable but also exhibits medicinal properties, including anti-inflammatory, antidiabetic, and anticancer effects. Moreover, the fiber derived from luffa finds extensive applications in various fields such as biotechnology and construction. However, luffa Fusarium wilt poses a severe threat to its production, and existing control methods have proven ineffective in terms of cost-effectiveness and environmental considerations. Therefore, there is an urgent need to develop luffa varieties resistant to Fusarium wilt. Single-plant GWAS (sp-GWAS) has been demonstrated as a promising tool for the rapid and efficient identification of quantitative trait loci (QTLs) associated with target traits, as well as closely linked molecular markers. RESULTS: In this study, a collection of 97 individuals from 73 luffa accessions including two major luffa species underwent single-plant GWAS to investigate luffa Fusarium wilt resistance. Utilizing the double digest restriction site associated DNA (ddRAD) method, a total of 8,919 high-quality single nucleotide polymorphisms (SNPs) were identified. The analysis revealed the potential for Fusarium wilt resistance in accessions from both luffa species. There are 6 QTLs identified from 3 traits, including the area under the disease progress curve (AUDPC), a putative disease-resistant QTL, was identified on the second chromosome of luffa. Within the region of linkage disequilibrium, a candidate gene homologous to LOC111009722, which encodes peroxidase 40 and is associated with disease resistance in Cucumis melo, was identified. Furthermore, to validate the applicability of the marker associated with resistance from sp-GWAS, an additional set of 21 individual luffa plants were tested, exhibiting 93.75% accuracy in detecting susceptible of luffa species L. aegyptiaca Mill. CONCLUSION: In summary, these findings give a hint of genome position that may contribute to luffa wild resistance to Fusarium and can be utilized in the future luffa wilt resistant breeding programs aimed at developing wilt-resistant varieties by using the susceptible-linked SNP marker.

Antifungal susceptibility of the clinical and environmental strains of Cryptococcus gattii sensu lato in Taiwan.

BACKGROUND: The rare occurrence of human cryptococcosis caused by Cryptococcus gattii sensu lato leads to difficulties in establishing the antifungal susceptibility profile between species of this potentially lethal pathogen, which may be crucial for treating cryptococcosis. OBJECTIVE: To establish an antifungal susceptibility profile of C. gattii s.l. in Taiwan. METHODS: A total of 104 environmental C. gattii s.l. strains (including multilocal sequence typing ST7, ST106, ST274, ST328, ST546, ST548 and ST630) and 21 previously collected clinical strains (including ST7, ST44, ST06, ST274, ST328 and ST329) were included in this study. We determined the minimum inhibitory concentrations (MICs) of six antifungal agents (itraconazole, fluconazole, voriconazole, posaconazole, flucytosine and amphotericin B) against environmental C. gattii s.l. strains and compared the antifungal susceptibility profiles of environmental strains with those of clinical strains. RESULTS: The antifungal susceptibility data demonstrated that the MICs of antifungal agents against environmental strains were comparable to those against clinical strains. Compared with strains of Cryptococcus deuterogattii, those of C. gattii sensu stricto were more susceptible to azoles and flucytosine. The differences in antifungal susceptibility between the strains of each sequence type (ST) were significant. Correlation analysis of MICs revealed cross-resistance between azoles in environmental strains of C. gattii s.l. Geographic differences in the antifungal susceptibility of C. gattii s.l. isolated from different cities in Taiwan were observed in this study. CONCLUSION: Clinical and environmental strains were indistinguishable in antifungal susceptibility. The antifungal susceptibility of C. gattii s.l. is associated with STs. Therefore, establishing an ST-oriented domestic antifungal susceptibility database may help treat C. gattii s.l.-induced cryptococcosis.

Comparative genomic and transcriptomic analyses of trans-kingdom pathogen Fusarium solani species complex reveal degrees of compartmentalization.

BACKGROUND: The Fusarium solani species complex (FSSC) comprises fungal pathogens responsible for mortality in a diverse range of animals and plants, but their genome diversity and transcriptome responses in animal pathogenicity remain to be elucidated. We sequenced, assembled and annotated six chromosome-level FSSC clade 3 genomes of aquatic animal and plant host origins. We established a pathosystem and investigated the expression data of F. falciforme and F. keratoplasticum in Chinese softshell turtle (Pelodiscus sinensis) host. RESULTS: Comparative analyses between the FSSC genomes revealed a spectrum of conservation patterns in chromosomes categorised into three compartments: core, fast-core (FC), and lineage-specific (LS). LS chromosomes contribute to variations in genomes size, with up to 42.2% of variations between F. vanettenii strains. Each chromosome compartment varied in structural architectures, with FC and LS chromosomes contain higher proportions of repetitive elements with genes enriched in functions related to pathogenicity and niche expansion. We identified differences in both selection in the coding sequences and DNA methylation levels between genome features and chromosome compartments which suggest a multi-speed evolution that can be traced back to the last common ancestor of Fusarium. We further demonstrated that F. falciforme and F. keratoplasticum are opportunistic pathogens by inoculating P. sinensis eggs and identified differentially expressed genes also associated with plant pathogenicity. These included the most upregulated genes encoding the CFEM (Common in Fungal Extracellular Membrane) domain. CONCLUSIONS: The high-quality genome assemblies provided new insights into the evolution of FSSC chromosomes, which also serve as a resource for studies of fungal genome evolution and pathogenesis. This study also establishes an animal model for fungal pathogens of trans-kingdom hosts.

Resistance to Fusarium oxysporum f. sp. luffae in Luffa Germplasm Despite Hypocotyl Colonization.

Fusarium wilt of Luffa, caused by Fusarium oxysporum f. sp. luffae, causes great losses in Luffa plants worldwide. In this study, 45 accessions of Luffa germplasm were used to determine their resistance to F. oxysporum f. sp. luffae isolates (FOLUST, FOLUSC, Fomh16, and Fol114) in two independent trials. In the first trial, only FOLUST was used to preliminarily identify resistant accessions. Nine accessions of Luffa acutangula and five of L. aegyptiaca were resistant to the FOLUST isolate. In the second trial, the other three isolates were then used to reevaluate the 14 resistant accessions. The results indicated that the 14 accessions were resistant to FOLUSC but exhibited variable resistance to the Fomh16 and Fol114 isolates. Eight accessions of L. acutangula and one accession of L. aegyptiaca were resistant to Fol114. Seven accessions of L. acutangula and one accession of L. aegyptiaca were resistant to Fomh16. Despite the lack of any symptoms, the F. oxysporum f. sp. luffae isolates were recovered from the hypocotyls of all resistant accessions at 28 days postinoculation, except for isolates FOLUSC and FOLUST on one accession (LA140). A high percentage (87.5%) of accessions collected from Bangladesh were identified as resistant, highlighting the effect of local adaptation on resistance. These results provide potentially valuable genetic resources for breeding programs to develop new varieties or rootstocks that could be beneficial for controlling soilborne diseases in different cucurbit crops and further investigating the mechanisms of resistance to F. oxysporum f. sp. luffae in Luffa plants.

First report of target spot disease of strawberry caused by Corynespora cassiicola in Taiwan.

Strawberry (Fragaria x ananassa Duch.) is an important crop worldwide. Tontonaka, Aroma and Benihoppe, are most popular cultivars in Taiwan, especially cv. Aroma is dominated in the market. In September 2021, the target spot outbreak occurred on the leaves of cv. Aroma and Benihoppe in Nantou County. In a greenhouse, the target spot incidents were estimated at 90-100% and 40-50% in Aroma and Benihoppe, respectively, and caused 2~5% plants lost. Between April and June of 2022, the target spot occurred in another greenhouse where the target spot incidents were 90% and 5-8% in Aroma and Benihoppe, respectively. Early symptoms were small and circular to irregular brown spot on the leaves with its diameter at 1-2 mm. Then the brown lesion expanded to 2-5 mm in diameter with pale green halo. Some lesions appeared with gray center, and 2-3 spots might merge into one lesion, and some lesions were surrounded with yellow tissues later. The round to oval brown spots were also observed on stems. Ten symptomatic leaves and stems each were collected for pathogen isolation. Pieces of tissue from the edge of the brown lesion on leaf were cut and disinfested with 0.6% NaOCl for 30 sec, and rinsed three times with sterile distilled water (SDW) followed by being placed on 2% water agar. The isolates obtained from symptomatic leaves/stems of Aroma and Benihoppe showed same colonies with 100% isolation rate. Isolates from cv. Benihoppe (Cos21-1) and Aroma (Cos21-2) were selected for further observation and tests. Colonies on potato dextrose agar exhibited gray aerial mycelium at 28 °C in dark after 7-day. Conidiophores were brown, single or in clustered, unbranched, 2 to 11 septa. Conidia were 5.6-6.7× 28.1-270.0 µm (n=50) in size with obclavate to cylindrical shape, 1 to 16 septa, and olivaceous to dark brown. Based on the morphology, two fungal isolates were identified as Corynespora cassiicola. Four regions, internal transcribed spacer (ITS), ß-tubulin, translation elongation factor (TEF), and actin, were used to confirm the two isolates. Sequences of ITS and ß-tululin shared 100% identity to ITS (MZ093622) and ß-tululin (MW961419) of C. cassiicola in GenBank. Sequences of TEF and actin shared 99.60% and 99.70% identity to C. cassiicola (MK882240 and FJ853005), respectively. For the pathogenicity test, the conidial suspension (1x105 spores/ ml) of Cos21-1 and Cos21-2 was sprayed on leaves of two-month-old strawberry cv. Benihoppe and Aroma without wounds, respectively. Three plants each with more than two leaves were spray-inoculated with the selected isolates whereas three plants with SDW as controls and the test was repeated once. Inoculated plants were covered with plastic bags in the greenhouse, then removed when the initial symptoms were observed on leaves 5 days after inoculation whereas symptoms on stems were observed within 7 days. Re-isolation of the pathogens from the symptomatic leaves/stems demonstrated that the pathogen was C. cassiicola. The leaf spot or target spot caused by C. cassiicola on strawberry has been reported in Mainland China and North America. To our knowledge, this is the first report of target spot disease of strawberry caused by C. cassiicola in Taiwan.

First Report of Fusarium incarnatum-equiseti Species Complex Causing Fruit Rot on Muskmelon in Taiwan.

Muskmelon (Cucumis melo L.) is an economically important fruit crop in Taiwan. In March 2020, the symptoms of fruit rot were observed in approximately 10% of mature muskmelon fruits in a field located in Wuri (24.043585, 120.657588), Taichung City, Taiwan. Symptoms including water-soaked lesions were initially observed on the lwer side of fruit, extending with time to cover most of the fruit area, and internal dissolution with white to brown mycelia on the surface was also observed. Ten rotted fruits were disinfested with 70% ethanol for 1 min followed by 1% NaOCl for 5 min, then rinsed three times with sterile distilled water (SDW). Fifteen sterilized symptomatic fruit fragments were cut into 1-cm3 pieces, placed on potato dextrose agar (PDA) amended with 35 mg/liter of streptomycin sulfate and incubated at 28°C in the dark for 1 week. Ten isolates with similar morphology were obtained and the representative isolate FOS-1 was characterized further. Single-spore isolates were used for morphological and molecular analyses. Isolates grown on PDA had dense, cottony white aerial mycelium, changed to light brown, and with the time yellowish-brown pigmentation appeared. Microconidia were ovoid, fusiform, or slightly curved, 0 to1 septate, and ranged between 7.9 to 16.5 × 2.8 to 3.5 µm. Macroconidia were 3 to 5 septate, with a slightly curved and tapering apical cell, and ranged between 18.7 to 35.1 × 3.3 to 4.1 µm. Spherical chlamydospores with thick walls were abundant and single, being produced in terminal or intercalary position. Based on morphological characteristics, the fungus was identified as Fusarium sp. (Leslie and Summerell 2006). PCR amplification and DNA sequencing were performed using primers ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999) to amplify the complete internal transcribed spacer (ITS) region and the partial translation elongation factor 1-alpha (TEF1-α) gene, respectively. The ITS and TEF1-α gene sequences of Isolate FOS-1 were deposited in GenBank database with acc. nos. MZ749694.1 and MZ782277.1, respectively. BLAST analysis showed 99.64% and 100% sequence identity with F. incanatum-equiseti species complex (FIESC) with MT563419.1 for ITS and MW034437.1 for EF-1α, respectively. BLAST analysis of TEF1-α gene sequence in FUSARIUM-ID database (Geiser et al. 2004), showed 99.31% sequence identity with FIESC (NRRL34070). Pathogenicity was confirmed by fulfilling Koch's postulates. Three healthy muskmelon fruit were disinfested using 70% ethanol for 30 s and 1% NaOCl for 5 min, and followed by three rinses with SDW. Then, the fruit were wounded using a sterile needle and inoculated with an 8 mm-mycelium agar plug. Three sites per fruit were inoculated, and three other fruits treated with mycelium-free PDA plugs served as the controls. The inoculated and control fruit were placed in a plastic box and incubated at 25°C under a 12 h photoperiod for 1 week. All inoculated fruit showed symptoms similar to those observed in the field, whereas no symptoms occurred on the controls. The fungus was re-isolated from the infected fruit, and identified as FIESC by the morphological and molecular methods described above. This pathogen could cause great losses in muskmelon. Members of the FIESC have been reported to cause leaf spot and fruit rot in muskmelon (Cao et al. 2019; Ismail et al. 2021). To our knowledge, this is the first report of the FIESC causing fruit rot of muskmelon in Taiwan. References: Cao, P., et al. 2019. Plant Dis.103:1768. Carbone, I., and Kohn, L. M. 1999. Mycologia. 91:553. Geiser, D.M., et al. 2004. Eur. J. Plant Pathol. 110:473. Ismail, S. I., et al. 2021. Plant Dis. 105:1197. Leslie, J. F., and Summerell, B. A. 2006. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, U.K. White, T. J., et al. 1990. PCR Protocols: A Guide to Methods and Applications Academic Press, San Diego, CA. 315.

First Report of Mango Leaf Blotch Caused by Pseudoplagiostoma mangiferae in Taiwan.

Mango (Mangifera indica L.) is an economically important tropical fruit in southern Taiwan. In February 2019, new leaf blotches distinct from anthracnose lesions were noticed on mango leaves in Meinong, Kaohsiung (N22°54'43.7" E120°32'59.3"). Symptoms were circular to irregular lesions with easily torn centers and were cream to light brown with dark brown margin on both leaf surfaces. Similar symptoms were observed on mango leaves in Yujing, Tainan (N23°07'31.3" E120°27'18.2") in July of the same year. We surveyed the disease incidence on 60 mango trees consisting of three cultivars, 'Irwin', 'Yu-win No.6' and a native cultivar in a commercial farm by randomly examining five shoots of each tree. The disease incidences of 'Irwin', the native cultivar and 'Yu-win No.6' were 25%, 37% and 73%, respectively. Diseased tissues from the two locations were surface sterilized and incubated on potato dextrose agar (PDA) for pathogen isolation. Seven isolates (Mgk3, TMg2-2.2, TMg3-1.2, TMg3-2.1, TMg4-1, TMg6-3, and TMg8-1.1) from different locations and cultivars were selected for further study. Pycnidia were produced on 7-day-old PDA cultures. Conidiogenous cells were hyaline and short cylindrical phialides. Conidia were hyaline, aseptate, thick-walled, 20-24 × 11-16 µm, and subcircular to ellipsoid with 1-2 large oil droplets and a markedly flat protruding hilum at the base. These morphological features presenting in the seven isolates were identified as a member of Pseudoplagiostoma (Cheewangkoon et al. 2010). Pathogenicity assays were conducted by the point inoculation method on 10 intact young leaves growing on 'Irwin' mango plants in a greenhouse at 20-25℃. Each leaf was inoculated at six points on the abaxial surface with point inoculation. Each point was inoculated with a 10 µl conidial suspension (106 conidia/ml) of isolate TMg 8-1.1. Sterilized water was used as control. Shoots with inoculated leaves were covered with translucent plastic bags for 2 days. At 7 days post-inoculation (dpi), 70% of conidia inoculated points (n = 60) displayed symptoms resembling the field symptoms, but sterilized water inoculated points (n = 60) did not. The six other isolates were inoculated on detached leaves by the point inoculation method. All inoculated leaves were maintained in humid containers at 25℃ with 12 h light/dark regime, and displayed similar lesions at 7 dpi. Fungi re-isolated from the symptomatic leaves showed the same morphological characteristics observed in the Pseudoplagiostoma originally isolated from diseased tissues. Internal transcribed spacer (ITS), TUB2 and LSU gene sequences of the seven isolates (ITS accession nos.: MN818659 to MN818665; TUB2 accession nos.: MW415921 to MW415927; LSU accession nos.: MN876849 to MN876855) were amplified with primer sets ITS4/V9G, T1/Bt-2b and LSU1Fd/LR5, respectively, and used for molecular identification (Cheewangkoon et al. 2010). The seven isolate were 95.8%, 99-100% and 99.8% identical to Pseudoplagiostoma mangiferae Dayarathne, Phookamsak & K. D. Hyde KUMCC 18-0179 (the ex-type strain) for the ITS gene (MK084824), TUB2 gene (MK084823) and LSU gene (MK084825), respectively. Phylogenetic analysis based on concatenated sequences of ITS and LSU genes was performed by the Maximum Likelihood method. All seven isolates were clustered in a well-supported clade with P. mangiferae KUMCC 18-0179 with 100% bootstrap value. Based on the pathogenicity and morphological characteristics, the pathogen was identified as P. mangiferae which was reported as a new species associated with mango leaf blight in Yunnan, China (Bezerra et al. 2019; Cheewangkoon et al. 2010; Crous et al. 2012; Crous et al. 2018; Phookamsak et al. 2019; Suwannarach et al. 2016). The newly emerging leaf blotch may become a prevalent disease of mango in future.

Molecular epidemiology and phylogenetic analyses of environmental and clinical isolates of Cryptococcus gattii sensu lato in Taiwan.

BACKGROUND: The rare occurrence of cryptococcosis caused by Cryptococcus gattii sensu lato (C. gattii s.l.) leads to the difficulties in studying the molecular epidemiology of this globally emerging disease. OBJECTIVES: To establish the molecular epidemiological profile of C. gattii s.l. in Taiwan, and understand the genetic relationship between locally endemic and global isolates. METHODS: A nationwide survey on environmental C. gattii s.l. in Taiwan was conducted from 2017 to 2019. The geographic distribution and molecular epidemiology based on multilocus sequence typing (MLST) data of the environmental isolates were compared with 18 previously collected clinical isolates. Phylogenetic analysis was performed to elucidate the genetic relationship between the global isolates and the isolates endemic to Taiwan. RESULTS: From a total of 622 environmental samples, 104 (16.7%) were positive for C. gattii s.l.. Seven sequence types were identified among the environmental isolates. The genetic population structure showed that the environmental and clinical isolates were closely linked by sequence types and geographical locations. Phylogenetic analysis revealed the association between the C. gattii s.l. isolates in Taiwan and those from South America and South Asia. The recombination test suggested that, in Taiwan, the C. gattii sensu stricto (C. gattii s.s). isolates undergo clonal reproduction and sexual recombination, whereas C. deuterogattii isolates were clonal. CONCLUSIONS: The molecular epidemiology of environmental C. gattii s.l. isolates is closely linked to the clinical isolates. Phylogenetic analysis of the environmental isolates provides an insight into the mechanisms underlying reproduction and dispersal of C. gattii s.l. in Taiwan.

Histopathology and quantification of green fluorescent protein-tagged Fusarium oxysporum f. sp. luffae isolate in resistant and susceptible Luffa germplasm.

Fusarium oxysporum f. sp. luffae (Folu) is a severe plant pathogen that causes vascular wilt and root rot in Luffa plants worldwide. A green fluorescent protein (GFP)-tagged isolate of Folu (Fomh16-GFP) was utilized to investigate the infection progress and colonization of Fomh16-GFP in resistant (LA140) and susceptible (LA100) Luffa genotypes. Seven days post-inoculation (dpi), it was observed that Fomh16-GFP had successfully invaded and colonized the vascular bundle of all LA100 parts, including the roots, hypocotyl, and stem. Pathogen colonization continued to increase over time, leading to the complete wilting of plants by 14-17 dpi. In LA140, the Fomh16-GFP isolate colonized the roots and hypocotyl vascular system at 7 dpi. Nevertheless, this colonization was restricted in the hypocotyl and decreased significantly, and no fungal growth was detected in the vascular system at 21 dpi. Thus, the resistant genotype might trigger a robust defense mechanism. In addition, while the pathogen was present in LA140, the inoculated plants did not exhibit any symptoms until 28 dpi. Quantitative PCR was utilized to measure the Fomh16-GFP biomass in various parts of LA100 and LA140 at different time points. The findings indicated a positive correlation between the quantity of Fomh16-GFP DNA and disease development in LA100. Alternatively, a high amount of Fomh16-GFP DNA was identified in the roots of LA140. Nonetheless, no significant correlations were found between DNA amount and disease progression in LA140. Aqueous extracts from LA140 significantly reduced Fomh16-GFP spore germination, while no significant reduction was detected using LA100 extracts.IMPORTANCEFusarium wilt of Luffa, caused by Fusarium oxysporum f. sp. luffae (Folu), causes great losses in Luffa plants worldwide. This study used a green fluorescent protein (GFP)-tagged isolate of Folu (Fomh16-GFP) to investigate the infection progress and colonization dynamics of Fomh16-GFP in the resistant and susceptible Luffa genotypes, which could be important in understanding the resistance mechanism of Folu in Luffa plants. In addition, our work highlights the correlations between DNA amount and disease progression in resistant plants using real-time PCR. We observed a positive correlation between the quantity of Fomh16-GFP DNA and disease progression in LA100, while no significant correlation was found in LA140. These results could be valuable to further investigate the resistance mechanism of Luffa genotypes against Folu. Gaining a better understanding of the interaction between Folu and Luffa plants is crucial for effectively managing Fusarium wilt and enhancing resistance in Luffa rootstock and its varieties.

Antifungal Activity of Cedrol from Cunninghamia lanceolate var. konishii against Phellinus noxius and Its Mechanism.

Phellinus noxius is a highly destructive fungus that causes brown root disease in trees, leading to decay and death. In Taiwan, five prized woods-Taiwania cryptomerioides, Calocedrus macrolepis var. formosana, Cunninghamia lanceolata var. konishii, Chamaecyparis formosensis, and Chamaecyparis obtusa var. formosana-are known for their fragrance and durability. This study aims to explore the anti-brown-root-rot-fungus activity of Cunninghamia lanceolata var. konishii (CL) essential oil (CLOL) and its primary components, while also delving into their mechanisms of action and inhibition pathways. The essential oil (CLOL) from CL wood demonstrated significant efficacy against P. noxius, with an inhibitory concentration (IC50) of 37.5 µg/mL. Cedrol, the major component (78.48%) in CLOL, emerged as a potent antifungal agent, surpassing the reference drug triflumizole. Further assays with cedrol revealed a stronger anti-brown-root-disease activity (IC50 = 15.7 µg/mL) than triflumizole (IC50 = 32.1 µg/mL). Scanning electron microscopy showed deformation and rupture of fungal hyphae treated with CLOL and cedrol, indicating damage to the fungal cell membrane. Cedrol-induced oxidative stress in P. noxius was evidenced by increased reactive oxygen species (ROS) levels, leading to DNA fragmentation, mitochondrial membrane potential reduction, and fungal apoptosis through the mitochondrial pathway. Gel electrophoresis confirmed cedrol-induced DNA fragmentation, whereas TUNEL staining demonstrated increased apoptosis with rising cedrol concentrations. Moreover, protein expression analysis revealed cedrol-triggered release of cytochrome c, activation of caspase-9, and subsequent caspase-3 activation, initiating a caspase cascade reaction. This groundbreaking study establishes cedrol as the first compound to induce apoptosis in P. noxius while inhibiting its growth through oxidative stress, an increase in mitochondrial membrane permeability, and activation of the mitochondrial pathway. The findings offer compelling evidence for cedrol's potential as an effective antifungal agent against the destructive brown root disease caused by P. noxius.

Endophytic Fungal Diversity in Cirsium kawakamii from Taiwan.

The endophytic fungal diversity of Cirsium kawakamii, a herb indigenous to Taiwan, was analyzed in this study. In addition, some fungal isolates were evaluated for the risk they pose as plant pathogens. In total, 1836 endophytic fungi were isolated from C. kawakamii from Hehuanjian, Puli Township, and Tatachia. They were classified into 2 phyla, 8 classes, 40 families, and 68 genera. Colletotrichum, Fusarium, Phomopsis, and Xylaria, (Ascomycota, Sordariomycetes) were the dominant genera. The genus accumulation curve (based on the bootstrap estimator) was non-asymptotic, with estimated richness significantly exceeding the richness captured by our sampling to date. Considering the collection time, the data indicated significant differences in the proportions of the C. kawakamii endophyte genus from Hehuanjan, Puli Township (across two seasons), and Tatachia. The Shannon and Gini-Simpson indices revealed variations in diversity, with C. kawakamii endophytes (Puli Township in winter) significantly reducing alpha diversity compared with other seasons and locations. Meanwhile, the Gini-Simpson index suggested that there were no significant differences in richness among the four sampling sites. The PCA results unveiled distinct community structures across different locations and seasons, explaining 46.73% of the total variation in fungal community composition significantly affected diversity and richness. In addition, a considerable number of Fusarium isolates exhibited harmful properties towards wheat, potatoes, and apples. It is postulated that these fungi belong to the Fusarium tricinctum species complex (FTSC).

Sensitivity of Colletotrichum gloeosporioides species complex (CGSC) isolated from strawberry in Taiwan to benzimidazole and strobilurin.

Colletotrichum gloeosporioides species complex (CGSC) is the major pathogen causing strawberry anthracnose in Taiwan. Benzimidazoles and strobilurins are common fungicides used to control strawberry anthracnose. A total of 108 CGSC isolates were collected from five major strawberry-producing areas in Taiwan. The half-maximal effective concentration (EC50) values of most CGSC isolates for benomyl (59 isolates), carbendazim (70 isolates), and thiabendazole (63 isolates) were higher than 500 µg a.i./mL. Strobilurin tests showed that the EC50 values of most CGSC isolates for azoxystrobin (66 isolates), kresoxim-methyl (42 isolates), and trifloxystrobin (56 isolates) were higher than 500 µg a.i./mL. However, most CGSC isolates were sensitive to pyraclostrobin at 100 µg a.i./mL. Fungicide tests indicated that CGSC isolates show multi-resistance to benzimidazoles and strobilurins. Benzimidazole-resistant isolates were associated with a point mutation in codon 198 of the ß-tubulin gene, and strobilurin-resistant isolates did not correspond with mutation in the cyt b gene or alternative oxidase activity.

Correction: Colonization of human opportunistic Fusarium oxysporum (HOFo) isolates in tomato and cucumber tissues assessed by a specific molecular marker.

[This corrects the article DOI: 10.1371/journal.pone.0234517.].

Colonization of human opportunistic Fusarium oxysporum (HOFo) isolates in tomato and cucumber tissues assessed by a specific molecular marker.

Fusarium oxysporum is a large complex cosmopolitan species composed of plant pathogens, human opportunistic pathogens, and nonpathogenic isolates. Many plant pathogenic strains are known based on host plant specificity and the large number of plant species attacked. F. oxysporum is an opportunistic pathogen in humans with a compromised immune system. The objectives of this study were: (1) to develop a specific marker to detect human opportunistic F. oxysporum (HOFo) isolates; (2) to determine whether or not HOFo isolates can colonize and cause disease symptoms in plants; and (3) to assess Taiwan isolates sensitivity to two agro-fungicides. The primer pair, Primer 5/ST33-R, specifically amplifying Taiwan and international reference HOFo isolates was developed and used to detect and assess the distribution of a Taiwan isolate in inoculated tomato plants and tomato and cucumber fruit. Taiwan HOFo isolate MCC2074 was shown to colonize tomato roots, hypocotyls, and cotyledons, but did not show any visible symptoms. Four days after surface inoculation of tomato and cucumber fruit with the same isolate, MCC2074 was detected in the pericarp and locular cavities of both tomato and cucumber fruit and in columella of tomato fruit. Three Taiwan HOFo isolates were found to be moderately sensitive to azoxystrobin and highly sensitive to difenconazole.

Two-step method for isolating Cryptococcus species complex from environmental material using a new selective medium.

Cryptococcosis is an opportunistic infection caused by the Cryptococcus species complex. An outbreak of cryptococcosis caused by Cryptococcus gattii (AFLP6/VGII) in North America has indicated the need for studies of this organism and its environmental niche. Difficulties in isolating the Cryptococcus spp. because of the overgrowth of filamentous fungi onto culture media and its low fungal population size under natural conditions limit studies of these pathogenic yeasts. We designed a selective medium that inhibits the growth of environmental filamentous fungi but does not inhibit that of Cryptococcus cells. After enrichment in acidified YPD media and inoculation onto selective media, Cryptococcus cells in brown-coloured colonies were isolated from environmental materials. This two-step method is useful for isolating environmental members of the Cryptococcus species complex, which is essential for further studies involving diversity and the microbe-environment relationship of this yeast.

BiOxCly/BiOmBrn/BiOpIq/GO quaternary composites: Syntheses and application of visible-light-driven photocatalytic activities.

Herein, the preparation of numerous bismuth oxychloride/bismuth oxybromide/bismuth oxyiodide/graphene oxide (BiOxCly/BiOmBrn/BiOpIq/GO) composites is reported. A facile hydrothermal method was employed to synthesize these photocatalysts, which had various GO contents. A total of 10 bismuth-oxyhalide composites were isolated and characterized using FE-SEM, XRD, FE-TEM, UV-Vis-DRS, FT-IR, EPR, HR-XPS, PL, and BET. The photocatalytic efficiencies of these 10 bismuth-oxyhalide composites were measured under visible-light irradiation by estimating the concentration of 2-hydroxybenzoic acid (HBA) degradation. The findings indicated that the rate constant order of the HBA degradations was BiOCl/BiOBr/BiOI/GO > Bi3O4Cl/Bi3O4Br/Bi4O5I2/GO > Bi12O17Cl2/Bi3O4Cl/Bi12O17Br2/ Bi7O9I3/GO > Bi12O17Cl2/BiOBr/BiOI/GO > Bi12O17Cl2/Bi12O17Br2/Bi7O9I3/Bi5O7I/GO > Bi3O4Cl/BiOBr/Bi3O4Br/Bi4O5I2 > Bi3O4Cl/BiOBr/BiOI > BiOCl/BiOBr/BiOI > Bi12O17Cl2/Bi5O7Br/Bi5O7I > GO. A maximum rate constant of 0.191 h-1 was reached for BiOCl/BiOBr/BiOI/GO, providing photocatalytic efficiency that was eight times higher than that of composite BiOCl/BiOBr/BiOI. We also proposed a photocatalytic mechanism demonstrating that O2-, h±, OH, and 1O2 are all essential for HBA degradation.

Ophiostoma breviusculum sp. nov. (Ophiostomatales, Ascomycota) is a new species in the Ophiostoma piceae complex associated with bark beetles infesting larch in Japan.

A new species of the Ophiostoma piceae-complex was isolated from bark beetles and the insect galleries of Larix kaempferi in Japan. This species was described as Ophiostoma breviusculum. The fungus was morphologically similar to O. piceae and O. quercus. However the average length of the perithecial necks and synnemata were shorter than for O. piceae and O. quercus. Synnemata morphological characteristics also differentiated O. breviusculum from the other species of the Ophiostoma piceae-complex isolated from conifers. Mating tests demonstrated that this fungus did not produce perithecia with O. floccosum, O. piceae and O. quercus. In phylogenetic trees using rDNA ITS O. breviusculum was placed in a clade with O. canum, O. piceae and O. subalpinum, but when using beta-tubulin it was placed into a separate clade.

Fungicide Sensitivity and Phylogenetic Relationship of Anthracnose Fungi Isolated from Various Fruit Crops in Japan.

Anthracnose diseases of fruit crops are mainly caused by Colletotrichum gloeosporioides and C. acutatum. In these Colletotrichum species, intra- and interspecific variation in fungicide sensitivity has been reported; however, the relationship between fungicide sensitivity and molecular phylogeny has not been analyzed. Fifty-one isolates from 10 fruit crops, acacia, and tea were tested for their sensitivities to thiophanate-methyl, diethofencarb, and iminoctadine-triacetate, and their internal transcribed spacer (ITS) and 5.8S regions of rDNA were analyzed. C. gloeosporioides isolates were divided into sensitive, less sensitive, intermediate resistant, or resistant to the three fungicides. In contrast, C. acutatum isolates were all less sensitive. In molecular phylogenetic analyses, C. gloeosporioides isolates fell into the same genetic group, whereas C. acutatum isolates were placed into two genetic groups. Although phylogenetic relationship was not closely related to fungicide sensitivity, the isolates of C. gloeosporioides most resistant to iminoctadine-triacetate were found in the same phylogenetic subgroup.

Controlled hydrothermal synthesis of BiOxCly/BiOmIn composites exhibiting visible-light photocatalytic degradation of crystal violet.

A series of BiOxCly/BiOmIn composites were prepared using autoclave hydrothermal methods. The composition and morphologies of the BiOxCly/BiOmIn composites were controlled by adjusting the experimental conditions: the reaction pH value, temperature, and KCl/KI molar ratio. The products were characterized using X-ray diffraction, scanning electron microscopy-electron dispersive X-ray spectroscopy, UV-vis diffuse reflectance spectroscopy, Brunauer-Emmett-Teller specific surface areas, cathodoluminescence, high-resolution transmission electron microscopy, and high-resolution X-ray photoelectron spectroscopy. The photocatalytic efficiencies of composite powder suspensions were evaluated by monitoring the crystal violet (CV) concentrations. In addition, the quenching effects of various scavengers indicated that the reactive O2(-) played a major role, and OH or h(+) played a minor role in CV degradation. The intermediates formed during the decomposition process were isolated, identified, and characterized using high performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry to elucidate the CV decomposition mechanism.

Determining the degradation efficiency and mechanisms of ethyl violet using HPLC-PDA-ESI-MS and GC-MS.

BACKGROUND: The discharge of wastewater that contains high concentrations of reactive dyes is a well-known problem associated with dyestuff activities. In recent years, semiconductor photocatalysis has become more and more attractive and important since it has a great potential to contribute to such environmental problems. One of the most important aspects of environmental photocatalysis is in the selection of semiconductor materials like ZnO and TiO2, which are close to being two of the ideal photocatalysts in several respects. For example, they are relatively inexpensive, and they provide photo-generated holes with high oxidizing power due to their wide band gap energy. In this work, nanostructural ZnO film on the Zn foil of the Alkaline-Manganese Dioxide-Zinc Cell was fabricated to degrade EV dye. The major innovation of this paper is to obtain the degradation mechanism of ethyl violet dyes resulting from the HPLC-PDA-ESI-MS analyses. RESULTS: The fabrication of ZnO nanostructures on zinc foils with a simple solution-based corrosion strategy and the synthesis, characterization, application, and implication of Zn would be reported in this study. Other objectives of this research are to identify the reaction intermediates and to understand the detailed degradation mechanism of EV dye, as model compound of triphenylmethane dye, with active Zn metal, by HPLC-ESI-MS and GC-MS. CONCLUSIONS: ZnO nanostructure/Zn-foils had an excellent potential for future applications on the photocatalytic degradation of the organic dye in the environmental remediation. The intermediates of the degradation process were separated and characterized by the HPLC-PDA-ESI-MS and GC-MS, and twenty-six intermediates were characterized in this study. Based on the variation of the amount of intermediates, possible degradation pathways for the decolorization of dyes are also proposed and discussed.