The Ninth Visual Object Tracking VOT2021 Challenge Results

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Isolation and Heterologous Expression of a Polygalacturonase Produced by Fusarium oxysporum f. sp. cubense Race 1 and 4

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

Isolation and Heterologous Expression of a Polygalacturonase Produced by Fusarium oxysporum f. sp. cubense Race 1 and 4

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

An Exo-Polygalacturonase Pgc4 Regulates Aerial Hyphal Growth and Virulence in Fusarium oxysporum f. sp. cubense race 4

Fusarium oxysporum f. sp. cubense race 4 (Foc4) causes Fusarium wilt that affects banana plants, and hence, the molecular mechanisms of its virulence need to be investigated. We purified an exo-polygalacturonase (exo-PG), Pgc4, from Foc4. Pgc4 has an apparent molecular weight of 50.87 kDa based on sodium dodecyl sulphate–polyacrylamide gel electrophoresis. We further performed its sequence analysis and biochemical characterization. The two pgc4 genes encoding Pgc4 from Foc4 and Foc1 were 1434 bp in length and encoded 477 amino acids with differences, due to some nucleotide differences between the two. The Km and Vmax values of Pgc4 purified from Foc4 were determined to be 0.45 mg/mL and 105.26 Units·mg·protein−1 ·min−1, respectively. The recombinant proteins, r-Foc1-Pgc4 and r-Foc4-Pgc4, were expressed and purified from Pichia pastoris and showed optimal Pgc4 activity at 55 °C and pH 4.0; both could induce tissue maceration and necrosis in the “Guangfen-1” and “Baxi” varieties of banana but to a different extent. Phenotypic assays and complementation analyses revealed that, compared to the wild-type, the generated Foc4Δpgc4 mutant strain showed a lower aerial hyphal growth, grew slower, and had a reduced virulence. Therefore, our results demonstrate the function of Pgc4 as a pathogenicity factor of Foc4

Virulence-Associated Genes of Calonectria ilicola, Responsible for Cylindrocladium Black Rot

The Cylindrocladium black rot caused by Calonectria ilicicola is a destructive disease affecting a broad range of crops. Herein, we study virulence-associated genes of C. ilicicolaCi14017 isolated from diseased peanut roots (Arachis hypogaea L.). Ci14017 was identified via phylogenetic analysis of the internal transcribed spacer region and standard Koch’s postulate testing. Virulence-associated genes were based on genome analyses and comparative analysis of transcriptome and proteome profiles of sensitive and resistant peanut cultivars. Ci14017 identified as C. ilicicola has a 66 Mb chromosome with 18,366 predicted protein-coding genes. Overall, 46 virulence-associated genes with enhanced expression levels in the sensitive cultivars were identified. Sequence analysis indicated that the 46 gene products included two merops proteins, eight carbohydrate-active enzymes, seven cytochrome P450 enzymes, eight lipases, and 20 proteins with multi-conserved enzyme domains. The results indicate a complex infection mechanism employed by Ci14017 for causing Cylindrocladium black rot in peanuts

Neodeightonia arengae Y. R. Xiong, Manawas., K. D. Hyde & Z. Y. Dong 2022, sp. nov.

Neodeightonia arengae Y.R. Xiong, Manawas., K.D. Hyde & Z.Y. Dong, sp. nov. (FIGURE 2) Index Fungorum number: IF558659 Facesoffungi number: FoF 10227 Etymology: Epithet refers to the host genus from which the fungus was isolated Holotype: ZHKU 21-0095 Saprobic on leaf of Arenga tremula (Arecaceae). Sexual morph: not observed. Asexual morph: Conidiomata 200–330 µm length × 140–190 µm diam. (x =250 × 160 µm, n=10), pycnidial, semi-immersed, solitary, globose, unilocular, black dots, a crack on the surface of the host where the conidiomata is located. Peridium 20–30 µm (x =21 µm, n=5), composed of thick-walled, brown-black cells of textura angularis, thin inner wall, almost reduced to conidiogenesis region. Conidiophores reduced to conidogenous cells. Conidiogenous cells hyaline, holoblastic, rough. Conidia 10–30 µm × 10–15 µm (x =21 × 11 µm, n=40), hyaline, subglobose to ellipsoid, aseptate, granular content, one to two large guttules, broadly rounded at both ends, thin-wall without mucilaginous. Culture characters: colonies on PDA reach 7 cm diam. at 28˚C after five days. Upper view wrinkled, filamentous, entire margin, flat, cloudy, fluffy for aerial hyphae, become gray-black with time, dense for aerial hyphae, reverse becomes black. Material examined: CHINA. South China Botanical Garden, Guangzhou, Guangdong Province, dead leaves of Arenga tremula (Blanco) Becc., 17 December 2020, Yinru Xiong, (holotype ZHKU 21-0095), living cultures ZHKUCC 21-0074 ex-holotype; ZHKUCC 21-0075, ZHKUCC 21-0076 ex-Paratype. Notes: Three isolates in the present study clustered together with the Neodeightonia species. The taxa from this study form two sister clades to N. palmicola (MFLUCC 10-0822) and N. planchoniae (MFLUCC 17-2427). Neodeightonia planchoniae is a sexual morph and it was characterized by sheaths around the ascospores, while N. arenge is an asexual morph and there was no sheath observed around the conidia. Neodeightonia arengae differs from N. palmicola conidia by the absence of the truncate base, while N. palmicola conidia sometimes appeared truncate at the base (Table 3). Neodeightonia arengae has one or two large guttules in conidia, while the conidia of N. palmicola lack guttules (Liu et al. 2010). The conidia of N. arengae have one morphological stage, hyaline, and aseptate, while the conidia of N. palmicola have two morphological stages initially hyaline. With age, conidia become cinnamon to sepia, forming one septum. Based on these polyphasic approaches we identified our isolates as novel Neodeightonia species.Published as part of Xiong, Yinru, Manawasinghe, Ishara S., Liao, Chunfang, Hyde, Kevin D. & Dong, Zhangyong, 2022, Neodeightonia arengae sp. nov., Botryosphaeriaceous taxa on Arenga tremula (Arecaceae) from Guangdong, China, pp. 130-140 in Phytotaxa 530 (2) on page 136, DOI: 10.11646/phytotaxa.530.2.1, http://zenodo.org/record/583261

Neomassaria khayae Y. H. Yang, C. F. Liao & Manawas. 2023, sp. nov.

Neomassaria khayae Y.H. Yang, C.F. Liao & Manawas., sp. nov. (FIGURE 2) Index Fungorum number: IF559792, Facesoffungi number: FoF 12675. Etymology: In reference to the plant genus Khaya, which fungus was collected. Holotype: ZHKU 22-0055 Saprobic on dead stem of Khaya senegalensis. Sexual morph: Ascomata 300–600× 400–600 μm (x̅ = 430 × 500 μm, n = 13), semi-immersed, solitary or gregarious, mostly globose, carbonaceous, uniloculate, dark brown to black, ostiolate, papillate. Ostioles central, short papillate. Peridium 20–100 μm wide (x̅ = 60 μm, n = 30), thick-walled, outer layer comprising 3–6 layers of brown to dark brown, thick-walled cells of textura prismatica, combined with the host tissues, inner layer comprising thin-walled, hyaline cells of textura subcircular. Hamathecium 1–2 μm wide (x̅ = 1.5 μm, n = 20), comprising branched, filamentous, septate, trabeculate pseudoparaphyses between the asci and the apex coated with a gelatinous substance. Asci 100–150 × 15–20 μm (x̅ = 122 × 12 μm, n = 30), 8-spored, cylindrical or clavate, bitunicate, fissitunicate, apically rounded, with an ocular chamber, with a bulbous short pedicel. Ascospores 25–35 × 2–8 μm (x̅ = 30 × 5 μm, n = 30), overlapping-biseriate, 1–2-septate, slightly constricted at the septa, fusiform to ellipsoid, hyaline, straight or slight flexuous, tapering from the middle to both ends, surrounded no mucilaginous sheath, smooth-walled, mature ascospores contain 2–4 distinct refractive oil globules. Asexual morph: Not observed. Culture characteristics: —Ascospores germinated on PDA within 24 hours at room temperature (25–28˚C), colonies on PDA reaching up to 20 mm diam. after three weeks at 25 °C. Above umbonate, colonies surface has black or grey irregular ring stripes, dense, slightly raised, surface not smooth, with cottony texture, the margin is irregular with crenate, brown in the centre, edges from inside to outside olivaceous black to pale brown. The reverse is dark brown to pale brown at the margin, and black in the centre. Material examined: — China, Guangdong Province, Guangzhou City, Haizhu District, Zhongkai University of Agriculture Engineering (23°6′32″ N, 113°16′37″ E, alt. 20 m), on the dead stem of Khaya senegalensis (Desr.) A. Juss. (Meliaceae), 27 July 2021, Y.H. Yang & C.F. Liao, FZL002 (ZHKU 22-0055, holotype), ex-type culture ZHKUCC 22-0104; living cultures ZHKUCC 22-0105, ZHKUCC 22-0106. Notes: —In the multigene (LSU, SSU, rpb2, and tef1 -α) phylogenetic analyses of our three isolates (ZHKUCC 22-0104, ZHKUCC 22-0105, and ZHKUCC 22-0106) formed a sister clade with N. hongheensis (KUMCC 21-0340 and KUMCC 21-0344) and N. alstoniae (AS 14/ MFLUCC 21-0213). Our isolation N. khayae have few difference with its closely related taxa by having short papillate, bigger ascomata than N. hongheensis (KUMCC 21-0340) and N. alstoniae (AS 14/ MFLUCC 21-0213), thicker peridium than N. hongheensis (KUMCC 21-0340) and N. alstoniae (AS 14/ MFLUCC 21-0213). Neomassaria khayae develop ascospores which are 1–2-septate, longer and narrower than N. hongheensis (KUMCC 21-0340) and N. alstoniae (AS 14/ MFLUCC 21-0213). In addition, morphological profiles of all Neomassaria species are provided in TABLE 1. The nucleotide differences between N. khayae and its phylogenetically related species are as follows; N. hongheensis (KUMCC 21-0344) LSU: 2.24% (19/847 bases), SSU: 0.88% (9/1019 bases), rpb2: 12.33% (128/1038), tef1 -α: 5.58% (50/896); N. alstoniae (AS 14/ MFLUCC 21-0213) LSU: 1.12% (17/846 bases), SSU: 1.21% (12/988 bases). Therefore, based on both morphological and phylogenetic evidence, N. khayae is described as a new species.Published as part of Yang, Yunhui, Liao, Chunfang, Manawasinghe, Ishara S., Farias, Antonio Roberto Gomes De & Dong, Zhangyong, 2023, Neomassaria khayae sp. nov., a novel pleosporalean species on Khaya senegalensis from Guangdong Province, China, pp. 65-77 in Phytotaxa 599 (1) on pages 69-70, DOI: 10.11646/phytotaxa.599.1.4, http://zenodo.org/record/798921