First Report of Fusarium oxysporum f. sp. apii race 4 Causing Fusarium Yellows on Celery in China.

Celery (Apium graveolens var. dulce), which belongs to the family Apiaceae, is one of the most widely cultivated vegetable crops in the world. During 2020 and 2021, celery plants with Fusarium yellows and root rot were observed in four approximately 0.3 ha sized fields located in Zhaili village (118°74'E, 36°67'N) of Shouguang city, Shandong province, China. Almost 50% of the plants were infected. Disease symptoms were comprised of wilting of outer-older leaves, overall stunted growth, rotted roots and stems, with eventual death of plants. A total of 7 diseased plants were collected from 4 fields and used for isolation and identification of the causal agent. Diseased root tissues were cut into 3 × 3 mm pieces from the edge of the rotting region, surface sterilized by soaking in 75% ethanol for 1 min, followed by three washes with sterile distilled water, and then placed on potato dextrose agar (PDA), and incubated at 28°C for 6 days in the dark. A total of 19 morphologically similar fungal isolates were obtained by single-spore subcultures. The colonies produced abundant, loosely floccose, white aerial mycelia and pale purple pigmentation on PDA. Microconidia were hyaline, zero to one septate, and ranged from 1.7 - 3.6 × 5.3 - 13.7 µm (n = 70). Macroconidia were falciform, hyaline, mostly four to five septate, and ranged from 2.2 - 4.2 × 12.4 - 45.4 µm in size (n = 70). These morphological characteristics were consistent with Fusarium oxysporum (Leslie and Summerell 2006). The genomic DNA of 19 isolates was extracted using the Plant Genomic DNA Kit (Tiangen, China). The translation elongation factor-1α (TEF-1α) and IGS rDNA regions were amplified with primers EF1/EF2 (O' Donnell et al. 1998) and iNL11/FoIGS-R (Epstein et al. 2017). BLAST analysis showed that 19 isolates were highly similar to Fusarium oxysporum, with 100% for TEF-1α (MN507109) and 99% for IGS rDNA (MT671188), respectively. The resulting 683-bp TEF-1α and 930-bp IGS rDNA sequences of isolate QC20091622 were deposited in GenBank with accession nos. ON260806 for TEF-1α and ON260805 for IGS rDNA, respectively. In a maximum-likelihood phylogenetic analysis based on TEF-1α and IGS rDNA sequences of F. oxysporum, using MEGAX software, isolate QC20091622 was grouped in the same clade with F. oxysporum f. sp. apii race 4, with a low bootstrap value of 54 between race 3 and race 4, indicating that the races are not distinguishable using only these two loci, as reported by Epstein et al (2022). Additional loci and other diagnostic methods are required to identify the race. Furthermore, the total DNA of 19 isolates was amplified by race-specific primers N4851-F/R (F. oxysporum f. sp. apii race 2) and N3875-2F/R (race 4), respectively (Epstein et al. 2017), and 187 bp product was amplified with primer pair N3875-2F/R, but none with primer pair N4851-F/R, so the isolates were identified as F. oxysporum f. sp. apii race 4. Pathogenicity of the 19 isolates was tested on potted celery plants (cv. 'Baimiao'). Ten healthy 6-week-old celery plants were inoculated by dipping the roots in a conidial suspension (107 conidia/mL) for 30 min. Control plants were dipped in sterile distilled water. The plants were then grown in a greenhouse maintained at 15°C (night)/26°C (day) and 90% relative humidity with natural daylight. The pathogenicity test was repeated twice. All inoculated plants started to wilt and developed root rot symptoms 14 days later, which were similar to those observed in the fields. The control plants remained healthy. F. oxysporum f. sp. apii race 4 was reisolated from the symptomatic roots, and their identity was confirmed by PCR, fulfilling Koch's postulates. To our knowledge, this is the first report of F. oxysporum f. sp. apii race 4 causing root rot on celery in China. F. oxysporum f. sp. apii race 4 has been a destructive pathogen in celery, prevention and control measures should be considered.

First Report of Fusarium solani Species Complex Causing Vascular Wilt on Cauliflower in China.

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.

First Report of Fusarium oxysporum Causing Wilt on Coriander in China.

Coriander (Coriandrum sativum L), which belongs to the family Apiaceae, is a medicinal and aromatic plant. In China, coriander is widely cultivated in several parts as a vegetable crop. During August 2019 to June 2020, wilting symptoms were observed on coriander (cv. 'Tiegan') in a commercial plantation, with disease incidence of approximately 25 to 40% in Xiajiawang village (118°88'E, 35°46'N) of Linyi city, Shandong province, China. Symptoms included wilting and leaf yellowing, plant stunting, root rot, and vascular discoloration of the stem bases and roots. A total of eight symptomatic plants were uprooted and collected from three fields. To determine the cause of the disease, symptomatic root tissues were excised, surface disinfected with 75% ethanol for 30s, followed by three washes with sterile distilled water, and then placed on potato dextrose agar (PDA) and incubated at 28°C for 6 days. In total, 10 cultures were obtained and purified by single-spore subcultures on PDA for morphological identification. The morphology of multiple colonies was consistent and originally white, later becoming light to dark purple in color with abundant aerial hyphae. Macroconidia were hyaline and falcate, straight to slightly curved, 3-4 septate, 27.86 to 34.23 × 4.07 to 6.13 µm (n = 30), with apical cells curved and basal cells foot-shaped. Microconidia were hyaline, oval or ellipsoid, 0-1 septate, with a flat base, measuring 5.67 to 9.37 × 3.66 to 5.40 µm (n = 30). These morphological characteristics resembled those of Fusarium oxysporum (Leslie and Summerell 2006). Genomic DNA was extracted from fungal mycelium using the Plant Genomic DNA Kit (Tiangen, China). The nuclear ribosomal internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α) and mitochondrial small subunit (mtSSU) genes were amplified with primer pairs ITS1/ITS4 (White et al. 1990), EF1Ha/EF2Tb (O' Donnell et al. 1998) and NMS1a/NMS2b (Li et al. 1994). The resulting ITS (550-bp), TEF1-α (681-bp) and mtSSU (692-bp) sequences of isolate QC20091601 were deposited in GenBank (accession nos. MW900439, MW692008 and MW711738, respectively). BLAST analysis demonstrated 100% identities to the ITS, TEF-1α and mtSSU sequences of F. oxysporum (MN856370.1, MN507110.1 and MN386808.1), respectively. According to the morphological and molecular identification, the fungus was identified as F. oxysporum. In the pathogenicity test, healthy coriander plants (cv. 'Tiegan') at the 4-true-leaf stage were inoculated by dipping the roots into a conidial suspension of 1 × 107 conidia/mL for 10 min. Plants dipped in sterile distilled water served as controls. All treated plants were placed in a greenhouse maintained at temperature 30°C and 80% relative humidity. Ten days later, inoculated plants developed typical symptoms of leaf yellowing, wilting and vascular discoloration, which were identical to those observed in the fields, whereas the control plants remained healthy. F.oxysporum was reisolated from the symptomatic roots, and its identity was confirmed by PCR with the primes described above, fulfilling Koch's postulates. To our knowledge, this is the first report of F. oxysporum as a pathogen on coriander in China. F. oxysporum is a destructive plant pathogen with an unusually broad host range and worldwide distribution, prevention and control measures should be taken in advance.