First report of Fusarium odoratissimum causing postharvest kiwifruit rot

Populations of Sclerotium rolfsii, the causal organism of Sclerotium root-rot on a wide range of hosts, can be placed into mycelial compatibility groups (MCGs). In this study, we evaluated three different molecular approaches to unequivocally identify each of 12 previously identified MCGs. These included restriction fragment length polymorphism (RFLP) patterns of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) and sequence analysis of two protein-coding genes: translation elongation factor 1α (EF1α) and RNA polymerase II subunit two (RPB2). A collection of 238 single-sclerotial isolates representing 12 MCGs of S. rolfsii were obtained from diseased sugar beet plants from Chile, Italy, Portugal, and Spain. ITS-RFLP analysis using four restriction enzymes (AluI, HpaII, RsaI, and MboI) displayed a low degree of variability among MCGs. Only three different restriction profiles were identified among S. rolfsii isolates, with no correlation to MCG or to geographic origin. Based on nucleotide polymorphisms, the RPB2 gene was more variable among MCGs compared with the EF1α gene. Thus, 10 of 12 MCGs could be characterized utilizing the RPB2 region only, while the EF1α region resolved 7 MCGs. However, the analysis of combined partial sequences of EF1α and RPB2 genes allowed discrimination among each of the 12 MCGs. All isolates belonging to the same MCG showed identical nucleotide sequences that differed by at least in one nucleotide from a different MCG. The consistency of our results to identify the MCG of a given S. rolfsii isolate using the combined sequences of EF1α and RPB2 genes was confirmed using blind trials. Our study demonstrates that sequence variation in the protein-coding genes EF1α and RPB2 may be exploited as a diagnostic tool for MCG typing in S. rolfsii as well as to identify previously undescribed MCGs.

Tobacco (Nicotiana tabacum L.) is an important cash crop in China, with an estimated production of 2.2 million tons every year (Berbeć 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).

In July 2010, fusarium wilt symptoms of tomato (Lycopersicon esculentum Mill.) plants were found in two commercial greenhouses in the Damyang area of Korea. Approximately 1% of 7,000 to 8,000 tomato plants were wilted and chlorotic in each greenhouse. The vascular tissue was usually dark brown and the discoloration extended to the apex. Fragments (each 5 × 5 mm) of the symptomatic tissue were surface-sterilized with 1% NaOCl for 1 min, then rinsed twice in sterilized distilled water (SDW). The tissue pieces were placed on water agar and incubated at 25°C for 4 to 6 days. Nine Fusarium isolates were obtained from four diseased plants, of which three isolates were identified as F. oxysporum based on morphological characteristics on carnation leaf agar medium and DNA sequences of the translation elongation factor 1-alpha (EF-1α) gene (2). Macroconidia were mostly 3- to 5-septate, slightly curved, and 28 to 53 × 2.8 to 5.2 μm. Microconidia were abundant, borne in false heads or short monophialides, generally single-celled, oval to kidney shaped, and 5 to 23 × 3 to 5 μm. Chlamydospores were single or in short chains. The EF-1α gene was amplified from three isolates by PCR assay using ef1 and ef2 primers (3), and the amplification products were sequenced. The nucleotide sequences obtained were deposited in GenBank (Accession Nos. KC491844, KC491845, and KC491846). BLASTn analysis showed 99% homology with the EF-1α sequence of F. oxysporum f. sp. lycopersici MN-24 (HM057331). Pathogenicity tests and race determination were conducted using root-dip inoculation (4) on seedlings of tomato differential cultivars: Ponderosa (susceptible to all races), Momotaro (resistant to race 1), Walter (resistant to races 1 and 2), and I3R-1 (resistant to all races). A spore suspension was prepared by flooding 5-day-old cultures on potato dextrose agar with SDW. Plants at the first true-leaf stage were inoculated by dipping the roots in the spore suspension (1 × 106 conidia/ml) for 10 min. Inoculated plants were transplanted into pots containing sterilized soil, and maintained in the greenhouse at 25/20°C (12/12 h). Twenty-four seedlings of each cultivar were arranged into three replications. An equal number of plants of each cultivar dipped in water were used as control treatments. Disease reaction was evaluated 3 weeks after inoculation, using a disease index on a scale of 0 to 4 (0 = no symptoms, 1 = slightly swollen and/or bent hypocotyl, 2 = one or two brown vascular bundles in the hypocotyl, 3 = at least two brown vascular bundles and growth distortion, 4 = all vascular bundles brown and the plant either dead or very small and wilted). All isolates caused symptoms of fusarium wilt on all cultivars except I3R-1, indicating that the isolates were race 3. The pathogen was reisolated from the discolored vascular tissue of symptomatic plants. Control plants remained asymptomatic, and the pathogen was not reisolated from the vascular tissue. Fusarium wilt of tomato caused by isolates of F. oxysporum f. sp. lycopersici races 1 and 2 has been reported previously; however, race 3 has not been reported in Korea (1). To our knowledge, this is the first report of isolates of F. oxysporum f. sp. lycopersici race 3 on tomato in Korea.

Cauliflower (Brassica oleracea var. botrytis L.), which belongs to the family Cruciferae, is a cool-season vegetable with green leaves around a large hard white head of flowers. China is the leading cauliflower and broccoli producing country in the world, with approximately 10.71 MT production (FAOSTAT 2019). During September 2018 to July 2019, wilting symptoms were observed on cauliflower in several commercial fields, with approximately 45% to 65% disease incidence in Shen county (115°48'E, 35°98N) of Liaocheng city, Shandong province, China. Plant stunting, leaves yellowing and wilting, and dark brown, hollow appearance of vascular stem tissues were the symptoms prominently observed. To isolate the causal organism, nine symptomatic tissues were collected and cut into small pieces (5 × 5 mm), disinfected in 75% ethanol for 30 s, rinsed three times in sterile water, transferred onto potato dextrose agar (PDA) medium. The plates were then incubated in air-conditioned room at 26°C with an artificial 12 h light-dark cycle provided by incandescent lamp. In total, 15 single-spore isolates were obtained and morphological characterization of 15 isolates was done on both PDA and carnation leaf agar (CLA; Leslie and Summerell 2006). The mycelia on PDA were initially white, fluffy, later becoming brown, and the underside of the colonies were light brown. Typical macroconidia were abundant on CLA. Macroconidia were hyaline, slightly curved, one to five septa, both ends were smooth, measuring 3.7 to 6.4 μm × 23.7 to 38.1 μm (n = 40). Microconidia were oval to cylindrical, hyaline, zero to one septate, measuring 2.0 to 4.1 × 4.3 to 10.3 μm (n = 40). Chlamydospores were terminal or intercalary, solitary or in pairs, globose to oval, thick wall, smooth or rough, 6.3 to 9.8 μm. Based on morphological characteristics, all of the 15 isolates were identified as Fusarium solani (Leslie and Summerell 2006). The isolates were further identified based on PCR amplification. The ITS, mtSSU, EF-1α and RPB2 genes were amplified using primers ITS1/ITS4, NSM1/NSM2 (Li et al. 1994), EF-1 Ha/EF-2Tb (O'Donnell et al. 1998) and RPB2-5F2/fRPB2-7cR (O'Donnell et al. 2008). BLAST analysis showed that 15 isolates were highly similar to F. solani species complex, with 100% similarity for ITS (AB470904.1), mtSSU (KF125009.1), EF-1α (KF372878.1), and RPB2 (MK048113.1), respectively. The sequences of isolate HYC1410080102 had been deposited in GenBank with accessions MT378292.1 for ITS, MT383122.1 for mtSSU, OK595059.1 for EF-1α and OK595060.1 for RPB2, respectively. Pathogenicity of the 15 isolates were conducted on 4-true-leaf seedlings cv. Jinsong by dipping the roots into a conidial suspension (107 conidia/mL) for 10 min. The conidial was prepared from 7-day old cultures grown on CLA at 26°C and suspended in sterilized water. Control plants were dipped in sterile distilled water. All treated seedlings were planted in 5.0 cm diameter plastic pots containing pasteurized soil matrix. Then the plants were kept in a greenhouse at 15°C (night)/26°C (day) and 80%RH with natural daylight. Twelve days later, brown lesions appeared on stem bases in all inoculated cauliflowers, and finally, the plants wilted, similar to those observed in the field. The control plants remained healthy. Re-isolation of the infected tissues showed same morphological characteristics of F. solani as the original isolates, which were verified using PCR. To our knowledge, this is the first report of F. solani causing cauliflower wilt in China and the world (Farr and Rossman 2021). F. solani is a destructive pathogen with a broad host range worldwide and is responsible for significant crop losses, prevention and control measures should be considered.

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.

Maize (Zea Mays L.) is one of the main crops in Ningxia Province, China, and stalk rot has become a serious disease of maize in this area. Infected plants showed softening of the stalks at lower internodes, which lodged easily and died prematurely during grain filling, and the pith tissue internally appeared to be disintegrating and slightly brown to reddish. In September 2018, symptomatic tissue was collected from seventeen locations in Ningxia. The incidence ranged from 5% to 40% in surveyed fields, reaching as high as 86% in certain plots. The discolored stalk pith tissues from the lesion region were cut into small pieces (approximately 0.5 × 0.2 cm), superficially disinfected with 75% ethanol for 1 min and rinsed three times with sterile water before plating on potato dextrose agar (PDA) medium with chloromycetin. The purified strains were obtained by single-spore separation and transferred to PDA and carnation leaf agar (CLA) medium. Morphological and molecular characteristics confirmed the presence of nine Fusarium species in these samples, including Fusarium graminearum species complex and Fusarium verticillioides. Four isolates of Fusarium nelsonii were recovered from samples collected in Shizuishan and Wuzhong. On PDA plates, the floccose to powdery, white to rose-colored aerial mycelia were produced and covered plates after 8 days of incubation, producing abundant mesoconidia and chlamydospores. Mesoconidia were fusiform or lanceolate until slightly curved with 0-3 septa, and chlamydospores were initially smooth and transparent, and became verrucous and light brown. Macroconidia produced in CLA were straight or curved and falcate, usually having 3-5 septa, with beak-shaped strongly curved apical cells and foot-shaped basal cells. Two isolates (SS-1-7 and ZY-2-2) were selected for molecular identification, and the total DNA was extracted using a fungal genomic DNA separation kit (Sangon Biotechnology, Shanghai, China). Sequence comparison of EF-1α (GenBank accession numbers MW294197 and MW294198) and RPB2 (Accession MW294176 and MW294177) genes showed 97% homology with the sequences of F. nelsonii reported in GenBank (accession MN120760 for TEF and accession MN120740 for RPB2). Pathogenicity tests with two isolates (SS-1-7 and ZY-2-2) were performed by individually inoculating five 10-leaf stage maize plants at between the 2nd and 3rd stem nodes from the soil level with 20 μl conidial suspension at a concentration of 106 conidia/ml as described by Zhang et al. (2016). Five maize plants inoculated with sterile water were used as controls. The inoculated plants were kept at 25 ± 0.5°C in the greenhouse with a photoperiod of 12 h. After 30 days, all plants inoculated with the conidial suspension formed an internal dark brown necrotic area around the inoculation site, whereas the control plants showed no symptoms. The pathogen was re-isolated from the necrotic tissue of the inoculated plants and identified by morphological characteristics as F. nelsonii. This species was first described by Marasas et al. (1998), and it is expanding its host range and has been isolated from sorghum, Medicago, wheat, and cucumber (Ahmad et al. 2020). The pathogen should be paid more attention owing to a serious risk of trichothecene and aflatoxin contamination (Astoreca et al. 2019; Lincy et al. 2011). To our knowledge, this is the first report of maize stalk rot caused by F. nelsonii in China.

Gerbera daisy, Gerbera jamesonii H. Bolus ex. Hooker, is an important flower grown globally. In September 2020, gerbera seedlings in a greenhouse farm in the region of Fujian, China, developed symptoms of severe wilting and stunting. The main stem exhibited reddish to light brown vascular discoloration. Approximately 30% of the 60,000 plants showed symptoms. To isolate the causal agent, necrotic tissue pieces (3×3 mm) from the symptomatic stem were surface-disinfected with 1% NaClO for 1 min and washed three times with sterile water. The disinfected pieces were dried and placed on potato dextrose agar (PDA) at 25°C in the darkness for 4 days inside a dark chamber. Reddish-white and floccose mycelia developed on PDA after 3 days incubation. Ten single-spored isolates were identified asFusarium kyushuensebased on morphological features (Aoki & O'Donnell, 1998). Hyaline and straight or slightly curved macroconodia were observed with 3 to 5 septate, 24.5 - 46.6 × 3.6 - 5.7 μm (n = 100). Microconidia were ellipsoidal to clavate, 0 to 1 septate, and 6.3 to 19.5 × 3.2 to 5.3 μm (n = 100). No chlamydospores were observed. In order to validate this result, partial RNA polymerase second largest subunit (RPB2) combined with translation elongation factor (EF-1α) gene regions were amplified and sequenced from three isolates with primers 5f2/11ar (Liu et al., 1999) and primers EF1/EF2 (Geiser et al.2004), respectively. Fusarium MLST analysis showed that the RPB2 (Genbank accession No. MZ130468, No. MZ130469, No. MZ130470) matched 99.72% (MH582170) to F. kyushuense, and the EF-1α (MZ130471, MZ130472, MZ130473) matched 99.84% (MH582297) to F. kyushuense in the Fusarium MLST. Besides, a phylogenetic analysis was conducted using the neighbor-joining algorithm based on the RPB2 and EF-1α gene sequences. The isolates clustered with F. kyushuense. To assess pathogenicity, the three molecularly identified isolates were used. The isolates were grown on carboxylmethyl cellulose (CMC) medium (carboxymethyl-cellulose (Sigma C-4888) 15.0 gram, NH4NO3 1.0 gram, KH2PO4 monobasic 1.0 gram, MgSO4·7H2O 0.5 gram, yeast extract 1.0 gram, distilled water filled to 1.0 liter) for sporulation. The roots of 12 healthy 30-day-old gerbera plants were inoculated by treating them with 10 mL of conidia suspension (1×106 conidial/mL). A group of 12 seedlings of the same age was treated with sterile water to serve as the control. Plants were grown in a glasshouse at 23 °C, relative humidity >70%, and 16 h light per day. Typical symptoms of wilt and discoloration of the vascular system in roots and stems developed within 10 days. Uninoculated plants remained healthy. Isolates were consistently re-isolated from the symptomatic stem and the recovered isolates were identified as F. kyushuense by amplifing the EF-1α gene. The assays were conducted twice. F. kyushuense has been reported to cause wilt and rot of tobacco (Wang et al., 2013), maize ears (Wang et al., 2014) and rice (Zhao et al., 2007) in China. To the best of our knowledge, this is the first report of F. kyushuense causing stem and root wilt on G. jamesonii. The disease must be considered in existing management practices.

Molecular phylogeny of Zygomycota based on EF-1α and RPB1 sequences: limitations and utility of alternative markers to rDNA

Fusarium pseudograminearum has previously been reported as a pathogen of wheat and barley in Henan Province of China (Li et al. 2012; Xu et al. 2017), but not of soybean (Glycine max L.). In 2017, soybean plants exhibiting symptoms of root rot characterized by brown lesions on the tap and lateral roots at the beginning pod (R3) growth stage were collected from 15 fields representing eight counties in Henan Province. The symptomatic root tissues were surface-sterilized with 70% ethanol for 50 s and rinsed in sterilized water three times. Small pieces of symptomatic tissue were then placed on potato dextrose agar (PDA) and incubated at 25°C for 5 days in the dark. A total of 132 Fusarium-like colonies were isolated, and hyphal tips were transferred to fresh PDA plates to obtain pure cultures. Single-conidia isolates were then produced by dilution plating conidia from pure cultures and then subculturing from colonies that developed from the single-conidia isolations. The average mycelial growth rate was 5.3 to 8.7 mm/day at 25°C on PDA, and the colonies produced aerial mycelium varying from rose to yellow white, and rose to burgundy pigment in agar. Macroconidia of the isolates were produced on carnation leaf agar (CLA) incubated under black light and observed to be abundant, relatively slender, almost straight, commonly 3- to 7-septate, averaged 38.6 × 4.1 μm, matching that reported for F. pseudograminearum (Aoki and O’Donnell 1999). No perithecia were observed. DNA of isolates YB17SN07 and YB17SN08 from Yuanyang County and isolate YB17SN09 from Yongcheng County was extracted using the CTAB method. The translation elongation factor 1 alpha (EF1-α) gene was amplified and sequenced (O’Donnell et al. 1998). Results of sequences were deposited in GenBank (accession nos. MG189598 to MG189600). BLASTn analysis of the EF1-α gene shared 99 to 100% similarity with those of F. pseudograminearum (HBZX1675 and CBS131261). The pathogen identity was further confirmed using species-specific PCR primers, Fp1-1 and Fp1-2 (Aoki and O’Donnell 1999). To test pathogenicity, inoculum was grown for 10 days at 25°C on 100 g of sterilized boiled wheat grain and sand (3:1 v/v) after infesting with a 1-ml spore suspension (10⁸ conidia/ml) of either isolate YB17SN07, YB17SN08, or YB17SN09. Five grams of this inoculum was placed 3 cm below the surface of autoclaved soil in 15 × 20 cm pots prior to planting pregerminated soybean seeds (cv. Wandou 35). Six plants were grown per pot, five pots were used per isolate, and the experiment was repeated three times. Noninoculated plants were grown in autoclaved soil amended with sterile wheat/sand mix and served as controls. Plants were grown in the greenhouse with a 14:10 h day/night cycle. After 42 days, the root system of all inoculated soybean plants exhibited dark brown lesions over the entire taproot. Noninoculated plants did not exhibit symptoms. The fungus was reisolated and identified from infected plants for each isolate as described above. This is the first report of F. pseudograminearum causing root rot on soybean in China. Considering that soybean is a major crop in China covering 7.8 million ha in 2017, root rot caused by F. pseudograminearum could develop into a disease with major economic impacts.

To determine a suitable DNA barcode for the genus Neonectria, the internal transcribed spacer rDNA, β-tubulin, EF-1α, and RPB2 genes were selected as candidate markers. A total of 205 sequences from 19 species of the genus were analyzed. Intra- and inter-specific divergences and the ease of nucleotide sequence acquisition were treated as criteria to evaluate the feasibility of a DNA barcode. Our results indicated that any single gene among the candidate markers failed to serve as a successful barcode, while the combination of the partial EF-1α, and RPB2 genes recognized all species tested. We tentatively propose the combined partial EF-1α and RPB2 genes as a DNA barcode for the genus. During this study, two cryptic species were discovered, based on the combined data of morphology and DNA barcode information. We described and named these two new species N. ditissimopsis and N. microconidia.

In 2011, common symptoms of grapevine dieback were frequently observed in 2- to 5-year-old table grape (Vitis vinifera L.) cvs. in four vineyards located in northern Tunisia. The symptoms included dead spur and cordons, shoot dieback, and sunken necrotic bark lesions, which progressed into the trunk resulting in the death of large sections of the vine. Longitudinal and transversal sections of cordons and spurs from symptomatic vines revealed brown wedge-shaped cankers of hard consistency. Twelve symptomatic samples from spur and cordons were collected, surface disinfected by dipping into 5% (v/v) sodium hypochlorite for 2 min, and small pieces from the edge of necrotic and healthy tissue were removed and plated onto potato dextrose agar (PDA) at 25°C in the dark. Based on colony and conidia morphological characteristics, isolates were divided in three species, named Diplodia seriata, Botryosphaeria dothidea, and Neofusicoccum luteum. D. seriata colonies were gray-brown with dense aerial mycelium producing brown cylindric to ellipsoid conidia rounded at both ends and averaged 22.4 × 11.7 μm (n = 50). B. dothidea colonies were initially white with abundant aerial mycelium, gradually becoming dark green olivaceous. Conidia were fusiform to fusiform elliptical with a subobtuse apex and averaged 24.8 × 4.7 μm (n = 50). N. luteum colonies were initially pale to colorless, gradually darkening with age and becoming gray to dark gray producing a yellow pigment that diffuses into the agar. Conidia were hyaline, thin-walled, aseptate, fusiform to fusiform elliptical, and averaged 19.8 × 5.5 μm (n = 50). Identity of the different taxa was confirmed by sequence analyses of the internal transcribed spacer (ITS1-5.8S-ITS2) region of the rDNA and part of the elongation factor 1-alpha (EF1-α) gene. BLAST analysis of sequences indicated that six isolates were identified as D. seriata (GenBank: AY259094, AY343353), one isolate as B. dothidea (AY236949, AY786319) and one isolate as N. luteum (AY259091, AY573217). Sequences were deposited in GenBank under accessions from KC178817 to KC178824 and from KF546829 to KF546836 for ITS region and EF1-α gene, respectively. A pathogenicity test was conducted on detached green shoots cv. Italia for the eight Botryosphaeriaceae isolates. Shoots were inoculated by placing a colonized agar plug (5 mm diameter) from the margin of a 7-day-old colony on fresh wound sites made with a sterilized scalpel. Each wound was covered with moisturized cotton and sealed with Parafilm. Control shoots were inoculated using non-colonized PDA plugs. After 6 weeks, discoloration of xylem and phloem and necrosis with average length of 38.8, 17.6, and 11.2 mm were observed from inoculated shoots with D. seriata, N. luteum, and B. dothidea, respectively, and all three fungi were re-isolated from necrotic tissue, satisfying Koch's postulates. Control shoots showed no symptoms of the disease and no fungus was re-isolated. In Tunisia, Botryosphaeria-related dieback was reported only on citrus tree caused by B. ribis (2), on Pinus spp. caused by D. pinea (4), on Quercus spp. caused by D. corticola (3), and on olive tree (Olea europea) caused by D. seriata (1). To our knowledge, this is the first report of D. seriata, B. dothidea, and N. luteum associated with grapevine dieback in Tunisia.

Fusarium dry rot of potato (Solanum tuberosum) is a postharvest disease caused by several Fusarium spp. Dry rot is managed primarily by reducing tuber bruising and promoting rapid wound healing. Dry rot symptomatic tubers were collected from Michigan seed lots in 2009 and 2010. The isolates may not have been exposed to fludioxonil because currently applications are restricted to seed not intended for seed production (3). Small sections were cut from the margins of necrotic regions with a scalpel, surface sterile in 10% sodium hypochlorite for 10 s, rinsed twice in sterile distilled water, and blotted with sterile filter paper. The tissue pieces were plated on half-strength potato dextrose agar (PDA) amended with 0.5 g/liter of streptomycin sulfate. The dishes were incubated at 23°C for 5 to 7 days. Cultures resembling Fusarium spp. were transferred onto water agar and hyphal tips from the margin of actively growing isolates were removed with a sterile probe and plated either on carnation leaf agar (CLA) or on half-strength PDA to generate pure cultures. Fusarium isolates were obtained and used for further studies. Among them, 54 were identified as Fusarium oxysporum and 23 as F. sambucinum. Identification was based on colony and conidial morphology on PDA and CLA, respectively. The identity was confirmed through DNA extraction followed by amplification and sequencing of the translation elongation factor (EF-1α) gene region. The Fusarium-ID v. (2) and the NCBI database were used to obtain the closest match to previously sequenced materials. Pathogenicity testing was done on disease-free potato tubers, cv. FL 1879. Tubers were surface sterilized for 10 min in 10% sodium hypochlorite and rinsed twice in distilled water. Three tubers per isolate were injected with 20 μl of a conidial suspension (106 conidia/ml) made from cultures grown on PDA for 7 days. Control tubers were injected with 20 μl of sterile distilled water. All tubers inoculated with F. sambucinum and F. oxysporum developed typical potato dry rot symptoms consisting of dry brown decay lesions. F. sambucinum and F. oxysporum were reisolated from all symptomatic tubers. An effective concentration for 50% reduction in growth (EC50) was determined for each F. sambucinum and F. oxysporum isolate for thiabendazole (TBZ), fludioxonil, and difenoconazole using the spiral gradient endpoint method (1). Sensitive and resistant F. sambucinum and F. oxysporum isolates were reported. Fifteen isolates of F. sambucinum and thirty-four of F. oxysporum were resistant to fludioxonil with EC50 greater than 130 mg/liter. The remainder was sensitive to fludioxonil with EC50 ranging from 0.8 to 4.9 mg/liter. To our knowledge, this is the first report of resistance to fludioxonil in isolates of F. sambucinum and F. oxysporum in Michigan. Fusarium insensitivity in laboratory studies may not translate directly to commercial production. This disparity may result from interactions not experienced in mixed populations or within a living host. There has been no compelling evidence to suggest that fludioxonil has failed to perform because of insensitivity to Fusarium. The occurrence of such isolated strains necessitates the development and registration of partner chemistries that can preempt any future concerns on lack of performance of products in use.

Panax notoginseng is one of the important economic crops under the forest, which is widely planted in Yunnan Province, China. In August of 2022, a survey in Xundian county (25º26' N, 12 103°7' E), was accomplished to verify the occurrence of wilt disease in P. notoginseng and understand its aetiology. The site is an underforest of organic P. notoginseng, covering an area of over 40 ha. Disease symptoms included severe stunting, leaf chlorosis, red or yellow stalks, and rotting roots. The entire plant gradually wilted and died with disease progression (Fig. 1). To identify the causal agent, we collected more than 30 wilted P. notoginseng plants and got the plant tissues from the symptomatic leaves, stalks, and roots. The tissues surface sterilised with 0.5% sodium hypochlorite for 2 min, followed by 75% alcohol for 1 min, and rinsed in sterilised water three times. Upon drying, samples were placed onto potato dextrose agar (PDA) incubated in the dark at 25°C (Bilgi et al. 2011). Isolates were then transferred to carnation leaf agar (CLA) to induce sporulation. Colonies on PDA were yellow, orange to red, with abundant fluffy aerial mycelia with a dark red pigment on the undersides; Colonies on CLA were orange to yellow (Fig. 2). Fusiform macroconidia and bottle-shaped conidiogenous cells were visible under a microscope. Microconidia were not observed. Macroconidia were measured as 18.5-40.5 μm × 3-4.7 μm (n = 60) (Fig. 3), and possessed 2 to 6 septa. These are similar to previously reported morphological characteristics of Fusarium graminearum (Shikur et al. 2018; Martinez et al. 2019). Cetyltrimethylammonium bromide rapid plant genome extraction kit-DN14 was used to obtain genomic DNA from two representative isolate, the ITS, TEF1 and RPB2 gene were amplified by Polymerase Chain Reaction using primers ITS5/ITS4 (White et al, 1990), EF1-983F/EF1-2218R (Rehner et al, 2005), bRPB2-6F/bRPB2-7.1R (Matheny et al, 2002), respectively. BLAST homology search for nucleotide sequences revealed > 99% similarity to F. graminearum ITS (550bp; MG274308, KU847854), TEF1 (1000bp; MH572248, MH572252) and RPB2 (1000bp; KT855203, KT855206) sequences. All sequences from this study were deposited in GenBank (OP617343 and OP617344 for ITS; OP930951 and OP930952 for TEF1; OP930953 and OP930954 for RPB2). In the phylogenetic tree, the isolates (SWFU 0000116, SWFU 0000117) clustered with the representative strains of F. graminearum. The morphology and multi-gene phylogenetic analysis indicated that the new isolate is F. graminearum. Koch's postulates were used to confirm that the symptoms in wilted P. notoginseng were attributable to F. graminearum. First, healthy leaves were gently wounded with a needle and sprayed with spore suspension (1.0 × 106 spores mL-1) in a hand sprayer (Martinez et al, 2019). All P. notoginseng plants were then replanted in pots with a diameter of 20 cm (1 plants/pot) filled with mixture of sterilised soil, and incubated at 25-27°C. The blank control comprised sterile cotton soaked in sterile water and inactivated mycelia sprayed on the leaves. After 7d of incubation, all inoculated leaves and stalks developed necrosis and developed pale red mycelia, while control plants remained symptomless (Fig. 4-5). The pathogen was successfully isolated from these inoculated plants and identified as F. graminearum. Koch's postulates were implemented. To the best of our knowledge, this is the first report from China with evidence of F. graminearum infecting P. notoginseng.

Cymbidium sinense (Jackson ex Andr.) Willd is a perennial terrestrial plant in the orchid family mainly distributed in China, Japan, India and Southeast Asia that occupies a strong position in the flower market due to its bright green leaves and fragrant flowers (Zhang et al. 2013). Cymbidium sinense is not only valued by people for its ornamental and economic value, but its roots have antiasthmatic medicinal properties (Ke et al. 2004). In August 2020, about 15% stem rot on two-year old C. sinense with varying severity was observed in five nursery gardens located in Enshi city (N 30° 16', E 109° 29'), Hubei province, China. Typical symptoms of C. sinense included roots and inner part of the pseudobulbs changing from white to brown and rotting. Leaves became brown and withered from bottom to top, and there was an obvious blight yellow halo at the junction of diseased and healthy tissue, which eventually caused the whole plant to wilt and die (Fig. 1d). To isolate the pathogen, a total of 15 leaf tissues from the disease-health junction (3 × 3 mm) from 5 individual plants (3 leaves/plant) with symptoms were surface sterilized with 75% ethanol for 30 s and 2% sodium hypochlorite (NaOCl) for 3 min. The sterilized tissue was rinsed three times with sterilized water, and then placed on potato dextrose agar (PDA) for incubation at 28°C in the dark for 5 days. Isolated colonies were subcultured by a hyphal tip protocol. Thirteen fungal isolates were obtained. Through preliminary pathogenicity tests, we found that ten isolates induced leaf blight. These ten isolates with pathogenicity showed similar morphological characteristics, with initial white-flocculent aerial mycelium that secreted a lavender pigment and produced colonies with an irregular edge after 3 days on PDA. The ten strains were cultured on PDA plates at 28℃ for 5 and 15 days to observe colony and conidial characteristics. The ten strains were identified as Fusarium based on morphological characteristics (Leslie and Summerell 2006). Strain ML0303 was selected for further identification. Macroconidia were falciform, hyaline, slightly pointed at both ends with two to four septa, 24.0 ± 5.6 µm × 4.7 ± 0.8 µm (n = 50). Microconidia were hyaline, oval, globose, with zero to one septum, 5.5 ± 1.3 µm × 2.2 ± 0.5 µm (n = 50) (Fig. 1c). Total genomic DNA of strain ML0303 was extracted with a CTAB protocol (Stenglein and Balatti 2006). The translation elongation factor (EF-1α), RNA polymerase II second largest subunit (RPB2) and β-tubulin (Tub2) genes were amplified respectively using primer pairs EF1/EF2, RPB2-5F2/RPB2-7cR and T1/T22 respectively (O'Donnell. et al. 2010, O'Donnell. et al. 1997). The EF-1α, RPB2 and Tub2 (accession numbers-MW719874, OL614838, OL689398, respectively) gene sequences were submitted to GenBank. EF-1α, RPB2 and Tub2 sequences of ML0303 showed 99.5% - 100% identity respectively with Fusarium oxysporum in the Genbank and FUSARIUM-ID databases. The multilocus sequence data was used to infer a phylogenetic tree via a Neighbor-joining (NJ), Maximum-likelihood (ML) and Maximum-Parsimony(MP) together with reference sequences from GenBank. The topology of the three trees was similar; only the NJ tree is presented here. Strain ML0303 and F. oxysporum formed a clade supported with high values (NJ/ML/MP: 96,95,97). The results indicated that the fungus was F. oxysporum based on the phylogenetic analysis and BLASTn queries. For pathogenicity tests, conidia of strain ML0303 were collected by rinsing PDA plates. Two-year-old C. sinense grown in plastic pots filled with sterilized autoclaved sandy loam soil were used for the tests. Three pots (two plants/pot) were included in each treatment. Spore suspensions (106spores/ml) of strain ML0303 were used to irrigate the stem-zone of the plants, and sterile water was used as control. The two treatments were placed in a greenhouse and incubated at 28±2℃ with a 14-hour light/10-hour dark cycle. The experiment was repeated twice. After three weeks, stem rot symptoms were observed on C. sinense inoculated with ML0303, that were the as same as observed in the nursery (Fig. 1e-h). No symptoms were observed on the negative control. Fusarium oxysporum was re-isolated from the infected plants to fulfill Koch's postulates. Partial EF-1α and RPB2 gene sequences were used for molecular identification. Members of the FOSC are notorious for causing many diseases, which includes stem rot of Sulcorebutia heliosa and root rot of Torreya grandis (Garibaldi et al. 2020; Zhang et al. 2016). To our knowledge, this is the first report of stem rot by F. oxysporum on C. sinense in China. The finding of this pathogen provides a clear target for stem rot control.

Root rot is an important disease of tea plants owing to its unobvious early symptoms and permanent damage (Huu et al. 2016). In 2019, 5% of tea plants displayed symptoms consistent with root rot in a tea plantation (28°09'N, 113°13'E) located in Changsha city, Hunan province of China. The symptoms of the diseased tea plants ranged from wilting leaves to entirely dead. The roots had black lesions and rot typical of this disease. Symptomatic roots were collected, washed with water and disinfected with 75% ethanol, then cut into pieces and sterilized with 0.1% mercuric chloride for 30 s, 75% ethanol for 1 min, and rinsed with sterile water five times. After drying on sterilized filter paper, root tissues were cultured on potato dextrose agar (PDA) medium at 25 oC for 7 days in the dark. Four isolates, CAGF1, CAGF2, CAGF3, and CAGF4 were purified by selecting single spores. All isolates were subjected to a pathogenicity test. A conidial suspension of each strain was collected at a concentration of 2×106 conidia/mL. For the pathogenicity test, two-year-old field grown tea plants were transplanted in plastic pots containing 240 g of the rice grain-bran mixture (inoculated with 4 mL of conidial suspension and cultured for 14 days) and 960 g of sterilized soil (Huu et al. 2016). The pots without inoculated mixture served as control group. All the pots were kept in illumination incubators at 25 oC and a 12L:12D photoperiod. The pathogenicity test for each strain was repeated three times with three repetitions. Only strain CAGF1 exhibited pathogenicity to tea plants. Symptoms appeared on the third day post inoculation (dpi) and gradually worsened by the 7 dpi. On the 14 dpi, most leaves had died and the roots were black and partially rotten, similar to field symptoms. The reisolated fungus from potted roots was identified as CAGF1 based on ITS region and colony morphology, while isolation was attempted, CAGF1 was not isolated from the control plants, which fulfilled Koch's postulates. On PDA, the colony center of CAGF1 was purple with white margin, while on carnation leaf agar (CLA) medium was white. On CLA medium, macroconidia have 0 to 3 septa, measured 19.1 μm to 41.2 μm × 4.2 μm to 5.4 μm (mean= 31.2 μm × 4.8 μm, n=30). The microconidia were measured as 6.7 μm to 12.8 μm × 2.4 μm to 4.9 μm (mean= 10.1 μm × 3.3 μm, n=30), with 0 to 1 septa. And the chlamydospores were measured as 6.0 to 9.7μm (mean= 7.7μm, n=30). Morphologically, strain CAGF1 was identified as Fusarium oxysporum (Leslie and Summerell 2006). Additionally, the genomic DNA of strain CAGF1 was extracted by cetyltrimethylammonium bromide (CTAB) method, the internal transcribed spacer (ITS), elongation factor 1 alpha (EF-1α) and second largest subunit of RNA polymerase II (RPB2) were amplified using the primers ITS1/ITS4 (White et al. 1990), EF-1/EF-2 (Geiser et al. 2004) and fRPB2-5F/fRPB2-7cR (Liu et al. 1999), respectively. Sequences were deposited in GenBank (ITS, OK178562.1; EF-1α, OK598121.1; RPB2, OP381476.1). BLASTn searches revealed that strain CAGF1 was 100% (ON075522.1 for ITS and JX885464.1 for RPB2) and 99.6% (JQ965440.1 for EF-1α) identical to Fusarium oxysporum species complex (FOSC). Based on phylogenetic analysis, the strain CAGF1 was identified as Fusarium cugenangense, belonging to FOSC. To our knowledge, this is the first report of F. cugenangense causing root rot of tea plants in China. The findings are important for the management of this root rot and the improvement of economic benefits of tea cultivation.