Diaporthe species are associated with Citrus as endophytes, pathogens, and saprobes worldwide. However, little is known about Diaporthe as endophytes in Citrus grandis in China. In this study, 24 endophytic Diaporthe isolates were obtained from cultivated C. grandis cv. “Tomentosa” in Huazhou, Guangdong Province in 2019. The nuclear ribosomal internal transcribed spacer (ITS), partial sequences of translation elongation factor 1-α (tef1), β-tubulin (tub2), and partial calmodulin (cal) gene regions were sequenced and employed to construct phylogenetic trees. Based on morphology and combined multigene phylogeny, eleven Diaporthe species were identified including two new species, Diaporthe endocitricola and D. guangdongensis. These are the first report of D. apiculata, D. aquatica, D. arecae, D. biconispora, D. limonicola, D. masirevicii, D. passifloricola, D. perseae, and D. sennae on C. grandis. This study provides the first intensive study of endophytic Diaporthe species on C. grandis cv. tomentosa in China. These results will improve the current knowledge of Diaporthe species associated with C. grandis. The results obtained in this study will also help to understand the potential pathogens and biocontrol agents and to develop a platform in disease management.
BackgroundFusarium wilt is an economically devastating disease that affects banana production. Although Cavendish banana cultivars are resistant to Fusarium oxysporum f.sp. cubense race 1 (FOC1) and maitain banana production after Gros Michel was destructed by race 1, a new race race 4 (FOC4) was found to infect Cavendish.ResultsAn exopolygalacturonase (PGC2) was isolated and purified from the supernatant of the plant pathogen Fusarium oxysporum f.sp. cubense race 4 (FOC4). PGC2 had an apparent Mr of 63 kDa by SDS-PAGE and 51.7 kDa by mass spectrometry. The enzyme was N-glycosylated. PGC2 hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by analysis of degradation products. To obtain adequate amounts of protein for functional studies between the PGC2 proteins of two races of the pathogen, pgc2 genes encoding PGC2 from race 4 (FOC4) and race 1 (FOC1), both 1395 bp in length and encoding 465 amino acids with a predicted amino-terminal signal sequence of 18 residues, were cloned into the expression vector pPICZaA and then expressed in Pichia pastoris strains of SMD1168. The recombinant PGC2 products, r-FOC1-PGC2 and r-FOC4-PGC2, were expressed and purified as active extracellular proteins. Optimal PGC2 activity was observed at 50°C and pH 5. The Km and Vmax values of purified r-FOC1-PGC2 were 0.43 mg.mL-1 and 94.34 units mg protein-1 min-1, respectively. The Km and Vmax values of purified r-FOC4-PGC2 were 0.48 mg.mL-1 and 95.24 units mg protein-1 min-1, respectively. Both recombinant PGC2 proteins could induce tissue maceration and necrosis in banana plants.ConclusionsCollectively, these results suggest that PGC2 is the first exoPG reported from the pathogen FOC, and we have shown that fully functional PGC2 can be produced in the P. pastoris expression system.
Pogostemon cablin is one of the well-known Southern Chinese medicinal plants with detoxification, anti-bacterial, anti-inflammatory, and other pharmacological functions. Identification and characterization of phytopathogens on P. cablin are of great significance for the prevention and control of diseases. From spring to summer of 2019 and 2020, a leaf spot disease on Pogostemon cablin was observed in Guangdong Province, South China. The pathogen was isolated and identified based on both morphological and DNA molecular approaches. The molecular identification was conducted using multi-gene sequence analysis of large subunit (LSU), the nuclear ribosomal internal transcribed spacer (ITS), beta-tubulin (β-tubulin), and RNA polymerase II (rpb2) genes. The causal organism was identified as Stagonosporopsis pogostemonis, a novel fungal species. Pathogenicity of Stagonosporopsis pogostemonis on P. cablin was fulfilled via confining the Koch's postulates, causing leaf spots and stem blight disease. This is the first report of leaf spot diseases on P. cablin caused by Stagonosporopsis species worldwide.
Fusarium wilt (Panama disease) caused by Fusarium oxysporum f. sp. cubense (FOC) represents a significant threat to banana (Musa spp.) production. Musa AAB is susceptible to Race 1 (FOC1) and Race 4 (FOC4), while Cavendish Musa AAA is found to be resistant to FOC1 but still susceptible to Race 4. A polygalacturonase (PGC3) was purified from the supernatant of Fusarium oxysporum f. sp. cubense race 4 (FOC4), which is the pathogen of Fusarium wilt. PGC3 had an apparent molecular weight of 45 kDa according to SDS-PAGE. The enzyme hydrolyzed polygalacturonic acid in an exo-manner, as demonstrated by analysis of degradation products. The Km and Vmax values of PGC3 from FOC4 were determined to be 0.70 mg·mL−1 and 101.01 Units·mg·protein−1·min−1, respectively. Two pgc3 genes encoding PGC3 from FOC4 and FOC1, both genes of 1368 bp in length encode 456 amino-acid residues with a predicted signal peptide sequence of 21 amino acids. There are 16 nucleotide sites difference between FOC4-pgc3 and FOC1-pgc3, only leading to four amino acid residues difference. In order to obtain adequate amounts of protein required for functional studies, two genes were cloned into the expression vector pPICZaA and then expressed in Pichia pastoris strains of SMD1168. The recombinant PGC3, r-FOC1-PGC3 and r-FOC4-PGC3, were expressed and purified as active proteins. The optimal PGC3 activity was observed at 50 °C and pH 4.5. Both recombinant PGC3 retained >40% activity at pH 3–7 and >50% activity in 10–50 °C. Both recombinant PGC3 proteins could induce a response but with different levels of tissue maceration and necrosis in banana plants. In sum, our results indicate that PGC3 is an exo-PG and can be produced with full function in P. pastoris.
The banana (Musa spp.) industry experiences dramatic annual losses from Fusarium wilt of banana (FWB) disease, which is caused by the fungus Fusarium oxysporum f. sp. cubense (FOC). Pisang Awak banana Fenza No. 1 (Musa spp. cv. Fenza No. 1), a major banana cultivar with high resistance to FOC race 4, is considered to be ideal for growth in problematic areas. However, Fenza No. 1 is still affected by FOC race 1 in the field. TR21 is an endophytic Bacillus subtilis strain isolated from orchids (Dendrobium sp.). Axillary spraying of banana plants with TR21 controls FWB, decreasing the growth period and increasing yields in the field. In this study, we established that TR21 increases root growth in different monocotyledonous plant species. By axillary inoculation, TR21 induced a similar transcriptomic change as that induced by FOC race 1 but also upregulated the biosynthetic pathways for the phytohormones brassinosteroid and jasmonate in Fenza No. 1 root tissues, indicating that TR21 increases FWB resistance, shortens growth period, and increases yield of banana by inducing specific transcriptional reprogramming and modulating phytohormone levels. These findings will contribute to the identification of candidate genes related to plant resistance against fungi in a non-model system and facilitate further study and exploitation of endophytic biocontrol agents.
Pleurotheciales is the largest order in Savoryellomycetidae with a large proportion of species known from freshwater habitats. In order to investigate the phylogenetic relationships of taxa within Pleurotheciales and contribute to their diversity, submerged wood was collected from freshwater habitats in China (Yunnan Province) and Thailand. Two dematiaceous, sporodochial hyphomycetes and one annulatascales-like ascomycete with unusual morphology as compared to extant ones were discovered. They were subjected to DNA-based phylogenetic analyses and the results revealed three distinct lineages in Savoryellomycetidae. This morpho-phylo taxonomic study supports the establishment of five novel taxa including two novel genera, Obliquifusoideum and Saprodesmium, and three novel species, Coleodictyospora muriformis, Obliquifusoideum guttulatum and Saprodesmium dematiosporum. Coleodictyospora muriformis and S. dematiosporum are placed in Pleurotheciales, while O. guttulatum is referred to Savoryellomycetidae genera incertae sedis. The phylogenetic relationships are also presented for Coleodictyospora and Pseudocoleodictyospora, which raises an intriguing taxonomic issue. These two genera are positioned in two different classes, viz Sordariomycetes and Dothideomycetes, although they are quite similar except for the presence of a conidial sheath. This study expands our knowledge of the fungal diversity of freshwater fungi, and also indicates that Pleurotheciales species are mostly found in freshwater habitats.
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