Characterization of Fusarium solani associated with tobacco (Nicotiana tabacum L.) root rot in Henan, China.

The aim of this study was to characterize the Fusarium solani species complex (FSSC) population obtained from tobacco roots with root rot symptoms using morphological characteristics, molecular tests, and assessment of pathogenicity. Cultures isolated from roots were white to cream with sparse mycelium on PDA with colony growth of 21.5 ± 0.5 to 29.5 ± 0.5 mm after 3 days. Sporodochia were cream on carnation leaf agar (CLA) and spezieller nährstoffarmer agar (SNA), and macroconidia formed in sporodochia were 3- to 6-septate, straight to slightly curved, with wide central cells, a slightly short blunt apical cell, and a straight to almost cylindrical basal cell with a distinct foot shape, ranging in size from 20.92 to 64.37 µm × 3.91 to 6.57 µm. Microconidia formed on CLA were reniform and fusiform with 0 or 1 to occasionally 2 septa, that formed on long monophialidic conidiogenous cells, with a size range of 5.99 to 32.32 µm × 1.76 to 5.84 µm. Globose to oval chlamydospores were smooth to rough-walled, 6.5 to 13.3 ± 0.37 µm in diameter, terminal or intercalary, single or in pairs, occasionally in short chains on SNA. Molecular tests consisted of sequencing and phylogenetic analysis of the translation elongation factor-1 alpha (EF-1α), RNA polymerase II largest subunit (RPB1), and second largest subunit (RPB2) regions. All the obtained sequences revealed 98.14%~100% identity to Fusarium solani in both Fusarium ID and Fusarium MLST databases. Phylogenetic trees of the EF-1α gene and concatenated three-loci data showed that isolates from tobacco in Henan grouped in the proposed group 5, which is nested within FSSC clade 3 (FSSC 5). Twenty-seven of the 28 isolates caused a root rot of artificially inoculated tobacco seedlings, with a disease index ranging from 15.00 ± 1.67 to 91.11 ± 2.22. Cross pathogenicity tests showed that three representative isolates were virulent to six species of Solanaceae and two of Poaceae, with disease indexes ranging from 6.12 ± 0.56 to 84.44 ± 0.00, indicating that these isolates have a wide host range. The results may inform control of tobacco root rot through improved crop rotations.

First report of Fusarium tricinctum Species complex causing root rot of tobacco in China.

Tobacco (Nicotiana tabacum L.) is an economically important crop in China. In June 2021, a root rot disease was observed on tobacco (cv. Yunyan99) in Lushi, Mianchi, and Luoning counties of western Henan, China. Diseased tobacco plants exhibited wilting with leaf chlorosis and root rot accompanied by purplish to brown vascular discoloration. The symptoms were observed in four surveyed fields, 57 ha in total, and disease incidence ranged from 21 to 56%. Five symptomatic plants with leaf chlorosis and root rot were randomly collected from each field for pathogen isolation. Tissue pieces from diseased roots were surface sterilized in 75% ethanol for 30 s then rinsed with sterile distilled water three times, air dried, and placed onto potato dextrose agar (PDA) medium. Five isolates, SL1, SL2, SL3, LN and KC, were purified by single-spore culturing. On PDA, colonies grew at a rate of 2-5 mm/day and produced abundant cottony, white to pink aerial mycelia and rose pigment on the reverse side of the culture plate. From 7-day-old cultures grown on carnation leaf agar (CLA), macroconidia were straight to subarcuate, with blunt and slightly hooked apical and basal cells, had three to four septa, measured 23.4 to 44.6×3.5 to 4.2 µm (n=30). Cylindrical, napiform or oval microconidia were one to two-celled, measuring 6.3 to 22.9×2.2 to 4.9 µm (n=30). Spherical chlamydospores were intercalary or terminal, in chains. Such characteristics resembled those of the Fusarium tricinctum species comples (FTSC; Batra and Lichtwardt 1962; Leslie and Summerell 2006). To confirm the morphological diagnosis, the genomic DNA of the isolates were extracted, the translation elongation factor 1-alpha (EF-1α), RNA polymerase I largest subunit (RPB1) and second largest subunit (RPB2) genes were amplified with primers EF1/EF2, F5/G2R and 5f2/7cr respectively (O'Donnell et al. 2010), and sequenced. Maximum likelihood analysis was carried out using MEGA 7. Sequences were 97.55% to 100% identical to corresponding DNA sequences of FTSC based on GenBank and Fusarium MLST BLASTn analysis, and deposited in GenBank (ON637268.1-ON637272.1, ON637275.1-ON637279.1, ON637282.1-ON637286.1). Based on the morphological characteristics and phylogenetic analysis, the isolates were identified as F. acuminatum (SL1, SL2, SL3 and LN; Senatore et al. 2021) and F. reticulatum (KC; Moreira et al. 2019). Koch's postulates were conducted to verify the pathogenicity of individual isolates. The four-leaf stage healthy tobacco seedlings (Yunyan99, n=30) were inoculated by pouring 20 mL conidial suspension (1×107 conidia/mL) around the rhizosphere. Control seedlings were inoculated with sterilized water (n=30). All the treatments were maintained under greenhouse conditions with a 12-h light/dark photoperiod at 25±0.5℃ and 70% relative humidity for 30 days. The assay was conducted three times. Foliage chlorosis and root rot were observed on the inoculated tobacco seedlings, whereas the control seedlings remained asymptomatic after 30 days. The pathogens were reisolated from the necrotic tissue from all inoculated seedlings and were identified by sequencing partial EF-1α and RPB2 genes. Fusarium tricinctum species complex are known as an important causal of cereals Fusarium Head Blight (FHB; Laraba, et al. 2022). In China, F. acuminatum can also infect herb plants and fruits, such as Angelica sinensis, Schisandra chinensis (Ma et al. 2022; Shen et al. 2021). To our knowledge, this is the first report of root rot on tobacco caused by FTSC members in China as well as the world. This finding expands the host range known for FTSC and will be helpful for developing effective control strategies of tobacco root rot.

The Characterization of the Tobacco-Derived Wild Tomato Mosaic Virus by Employing Its Infectious DNA Clone.

Viral diseases of cultivated crops are often caused by virus spillover from wild plants. Tobacco (N. tabacum) is an important economic crop grown globally. The viral pathogens of tobacco are traditional major subjects in virology studies and key considerations in tobacco breeding practices. A positive-strand RNA virus, wild tomato mosaic virus (WTMV), belonging to the genus potyvirus in the family potyviridae was recently found to infect tobacco in China. In this study, diseased tobacco leaf samples were collected in the Henan Province of China during 2020-2021. Several samples from different locations were identified as WTMV positive. An infectious DNA clone was constructed based on one of the WTMV isolates. By using this clone, we found that WTMV from tobacco could establish infections on natural reservoir hosts, demonstrating a possible route of WTMV spillover and overwintering in the tobacco field. Furthermore, the WTMV infection was found to be accompanied by other tobacco viruses in the field. The co-inoculation experiments indicate the superinfection exclusion (SIE) between WTMV and other potyvirus species that infect tobacco. Overall, our work reveals novel aspects of WTMV evolution and infection in tobacco and provides an important tool for further studies of WTMV.

First Report of Tobacco Root Rot Caused by Fusarium falciforme in China.

China is the largest producer of tobacco (Nicotiana tabacum L.) in the world with an estimated production of 2.4 million ton per year (Berbec and Matyka 2020). In June 2021, a root disease was observed on tobacco in three surveyed counties (Xiangcheng, Linying and Jiaxian) in central Henan. Diseased plants exhibited leaf chlorosis and brown to purplish vascular discoloration of the taproot and lateral roots. Approximately 10 to 15% of the plants were symptomatic in the nine fields surveyed, representing 60 ha in total. Root segments (0.5 to 1 cm) from ten diseased plants were surface sterilized in 75% ethanol for 30 s followed by rinsing with sterile distilled water three times. Thirty air dried root pieces were placed on potato dextrose agar (PDA) and incubated at 25℃ in the dark for 2 days. Typical Fusarium spp. colonies were obtained from all root samples. Ten pure cultures were obtained by single-spore culturing (Yz01 to Yz10). Colonies on PDA showed abundant white to cream aerial mycelia with a yellowish-brown center on the reverse side after 7 days, and an average growth rate of 5 mm/day. From 7-day-old cultures grown on carnation leaf agar (CLA), macroconidia had three to four septa, were falciform, with blunt apical cells and slightly hooked basal cell, and measured 20 to 41×3-6.5 µm (n=50). Spherical conidia clusters were formed at the apex of the conidiophores. Abundant reniform and cylindrical microconidia were one to two-celled, with apexes rounded, measuring 7 to 15×2 to 5 µm (n=50). The roughly spherical chlamydospores were intercalary or terminal, single or in chains, and rough walled. Such characteristics were consistent with the Fuarium solani species complex (FSSC) (Leslie and Summerell 2006). The translation elongation factor 1-alpha (EF1-α) gene of the ten cultures was amplified with primers EF1/EF2 (O'Donnell et al. 1998), and sequenced. Maximum likelihood analysis was carried out using the EF1-α sequences of the ten cultures (Kumar et al. 2016). The RNA polymerase I largest subunit (RPB1) and second largest subunit (RPB2) genes of the cultures were amplified with primers F5/G2R and RPB2F/R respectively (O'Donnell et al. 1998, 2010), and sequenced. The EF1-α, RPB1 and RPB2 sequences (GenBank accession nos. ON186742.1-ON186751.1, ON241133.1-ON241148.1, ON324054.1-ON324057.1) were 99.4 to 100% identical to the corresponding DNA sequences of Fusarium falciforme based on FUSARIUM-ID BLASTn analysis. Morphological and molecular results confirmed this species as F. falcifome (Díaz-Nájera et al. 2021; Velarde-Félix et al. 2022). Pathogenicity tests were performed in tobacco seedlings grown on autoclaved soil. Healthy six-leaf stage tobacco seedlings (n=30; Zhongyan 100) were inoculated by placing 7-days old wheat seed (15 seeds per plant) infested with the representative culture Yz07 around the root. Thirty seedlings inoculated with sterile wheat seeds served as controls. All the plants were maintained in a growth chamber at 25±0.5℃ and 70% relative humidity. The assay was conducted three times. Typical symptoms of foliage chlorosis and root browning were observed 7 to 14 days after inoculation for all the 90 inoculated seedlings. Fifteen diseased seedlings were randomly selected for tissue isolation, and F. falciforme was reisolated from the 15 seedlings and showed the same morphology and EF1-α gene sequence as the original isolate. Control plants remained asymptomatic and no pathogen was recovered. The results showed that F. falciforme can cause root rot of tobacco. F. falciforme was reported to cause tobacco wilt and root rot in Northwestern Argentina (Berruezo et al. 2018); however, this is the first report of F. falciforme causing root rot of tobacco in China. This species was previously reported in China affecting Weigela florida (Shen et al. 2019) and Dioscorea polystachya (Zhang et al. 2020), showing that F. falciforme has a broad host range in this country. These results may inform control tobacco root rot through improve crop rotations. Funding: Funding was provided by the Science and Technology Project of Henan Provincial Tobacco Company (2020410000270012), Outstanding Youth Science and Technology Fund Project of Henan Academy of Agricultural Sciences (2022YQ09) and Science and Technology Innovation Team project of Henan Academy of Agricultural Sciences (2022TD26). References: Berbec, A. K., and Matyka, M. 2020. Agric. 10:551. Berruezo, L. A., et al. 2018. Eur. J. Plant. Pathol. 151:1065. Díaz-Nájeraet, J. F., et al. 2021. Plant Dis. 105:710. Douriet-Angulo, A., et al. 2019. Plant Dis. 103:11. Kumar, S., et al. 2016. Mol. Biol. Evol. 33:1870. Leslie, J. F., and Summerell, B. A., eds. 2006. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA. O'Donnell, K., et al. 1998. PNAS. 95:2044. O'Donnell, K., et al. 2010. J. Clin. Microbiol. 48:3708. Vega-Gutierrez, T. A., et al. 2018. Plant Dis. 103:1. Velarde-Félix, S., et al. 2022. Plant Dis. 106:329. Zhang, X., et al. 2020. Plant Dis. 104:5. The author(s) declare no conflict of interest. Keywords: tobacco root rot, Fusarium falciforme, China.

Complete genome sequence of a novel mitovirus from the phytopathogenic fungus Fusarium oxysporum.

Fusarium oxysporum is a cosmopolitan plant pathogen that causes fusarium wilt and fusarium root rot in many economically important crops. There is still limited information about mycoviruses that infect F. oxysporum. Here, a novel mitovirus tentatively named "Fusarium oxysporum mitovirus 1" (FoMV1) was identified in F. oxysporum strain B2-10. The genome of FoMV1 is 2,453 nt in length with a predicted AU content of 71.6% and contains one large open reading frame (ORF) using the fungal mitochondrial genetic code. The ORF putatively encodes an RNA-dependent RNA polymerase (RdRp) of 723 aa with a molecular mass of 84.98 kDa. The RdRp domain of FoMV1 shares 29.01% to 68.43% sequence identity with the members of the family Mitoviridae. Phylogenetic analysis further suggested that FoMV1 is a new member of a distinct species in the genus Mitovirus.

First Report of Root Rot of Tobacco Caused by Fusarium brachygibbosum in China.

Tobacco (Nicotiana tabacum L.) is an important cash crop in China, with an estimated production of 2.2 million tons every year (Berbec and Matyka, 2020). In June 2020, a root rot disease was observed on tobacco (cv. Zhongyan 100) in four surveyed counties (Mianchi, Lushi, Duguan and Lingbao) in Sanmenxia. Diseased plants exhibited leaf chlorosis and purplish to brown vascular discoloration of stem, taproot and lateral roots. The disease incidence ranged from 15% to 40% in 11 surveyed fields, 36.7 ha in total. Twenty five diseased tissues were surface sterilized in 75% ethanol and placed on potato dextrose agar (PDA) medium. Fifteen single-spore isolates were obtained from 25 diseased tissue samples. All cultures growing on PDA had white colonies with abundant aerial mycelia initially, turning into yellow to orange in the center and produced red pigmentation after seven days of growth. The 7-day-old cultures grown on carnation leaf agar (CLA) produced macroconidia that were curved with 3-5 septa, had wide central cells, slightly pointy apex, and measured 17.0-45.9 µm long×3.0-4.6 µm wide (n=50). The microconidia formed on CLA were slightly curved, ovoid with zero to two septa, measuring 5.4-15.5 µm long×2.0-3.2 µm wide (n=50). Spherical chlamydospores (7.58-13.52 µm; n=50) were terminal or intercalary, single or in chains. Such characteristics were typical of Fuarium brachygibbosum (Tirado-Ramírez et al. 2018). DNA from one representative single-spore isolate (MC1) was extracted, and the translation elongation factor 1-alpha (EF1-α), RNA polymerase I largest subunit (RPB1) and second largest subunit (RPB2) genes were amplified with primers EF1/EF2, F5/G2R and RPB2F/R respectively (O'Donnell et al. 1998, 2010), and sequenced. Sequences were submitted to GenBank under accession numbers MT947796 (EF1-α), MW679536 (RPB1) and MW430664 (RPB2). The consensus sequences showed 99.70%, 99.94% and 100% identity to the sequences of F. brachygibbosum strain NRRL 34033 (accession no. GQ505418.1, HM347172.1 and GQ505482.1, Wang et al 2021). Morphological and molecular results confirmed this species as F. brachygibbosum (Al-Mahmooli, et al., 2013, Rentería -Martínez, et al., 2018). Pathogenicity tests were performed on tobacco seedlings grown on autoclaved tobacco specific substrate (Tobacco specific matrix, Ainong Biotechnology Co. Ltd, China). Healthy six-leaf stage tobacco seedlings (n=30; Zhongyan 100) were inoculated by placing 7-days old wheat seed (15 seeds per plant) infested with MC1 around the root. Thirty seedlings inoculated with sterile wheat seeds served as controls. All the plants were maintained in a growth chamber at 25±0.5℃ and 70% relative humidity. The assay was conducted three times. Typical symptoms of foliage chlorosis and root browning were observed 7-14 days after inoculation. The pathogen was reisolated from the necrotic tissue from all inoculated seedlings and was identified by sequencing partial EF1-α and RPB2 genes. Control plants remained asymptomatic and no pathogen was recovered from the control plants. Fusarium brachygibbosum is known as a pathogen of grains and cash crops in China (Shan, et al., 2017, Xia, et al., 2018). To our knowledge, this is the first report of F. brachygibbosum causing root rot on tobacco. We believe that our results will help to better understand rhizome fungal diseases affecting tobacco production in China. Acknowledgements: Funding was provided by the Science and Technology Project of Henan Provincial Tobacco Company (2020410000270012), Independent Innovation Project of Hennan Academy of Agricultural Sciences (2020ZC18) and Research and Development project of Henan Academy of Agricultural Sciences (2020CY010). References: Al-Mahmooli, I. H., et al. 2013. Plant Dis. 97:687; https://doi.org/10.1094/PDIS-09-12-0828-PDN Berbec A. K. and Matyka M. 2020. Agric. 10(11), 551; https://doi.org/10.3390/agriculture10110551 O'Donnell, K., et al. 1998. P. Natl. Acad. Sci. USA. 95(5):2044-2049; https://doi.org/10.1073/pnas.95.5.2044 O'Donnell, K., et al. 2010. J. Clin. Microbiol. 48(10)3708-3718; https://doi.org/10.1128/JCM.00989-10 Rentería -Martínez M.E., et al. 2018. Mex. J. of Phytopathol. 36(2):1-23; https://doi.org/10.18781/R.MEX.FIT.1710-1 Shan, L. Y., et al. 2017. Plant Dis. 101:837; https://doi.org/10.1094/PDIS-10-16-1465-PDN Tirado-Ramírez, M. A., et al. 2018. Plant Dis. 103; https://doi.org/10.1094/PDIS-04-18-0710-PDN Wang, S., et al. 2021. Plant Dis. 2021 Jan 6. doi: 10.1094/PDIS-05-20-0941-PDN. Epub ahead of print. PMID: 33406862. Xia, B., et al. 2018. Plant Dis. 102(11):2372; https://doi.org/10.1094/PDIS-12-17-1939-PDN The author(s) declare no conflict of interest.