RESUMO
In June 2021, a disease of stem and leaf rot was observed on lily cultivar 'Tresor' with approximately 20% disease incidence in fields at Huaiyin District (119°04'N, 33°63'E) of Huaian County, Jiangsu Province. The roots and bulbs of symptomatic plants were brown and rotten, with sunken lesions. Symptomatic plants showed short, discolored leaves, and eventually lead to stem wilt and death of the whole plants (Fig. 1A and Fig. 3C). To isolate the pathogen, necrotized plant tissues were surface sterilized with 2% sodium hypochlorite for 2 min followed by 70% ethanol for 30 s and rinsed with sterile water. About 4 mm × 4 mm of diseased tissues were placed on potato dextrose agar (PDA) followed by incubation at 25°C in the dark for 5 days. The pure cultures were obtained by the hyphal-tip method. A total of four fungal isolates with similar colony characteristics were recovered. To determine the identity of the four isolated fungal isolates, genomic DNA was extracted using the method previously described (Khan et al. 2021), the sequences of the internal transcribed spacer (ITS), the translation elongation factor 1α (TEF1) and the RNA polymerase II beta subunit (RPB2) genes were analyzed with primers ITS1/ITS4 (White et al. 1990), EF1/ EF2 (O'Donnell et al. 1998), and 5F2/7cR (Reeb et al. 2004), respectively. The three gene sequences of four isolates showed 99.9 %-100% identities. The531 bp (ITS), 699 bp (TEF1), and 900 bp (RPB2) sequences of a representative isolate (JH-37) were deposited in GenBank with acce. nos. OR195729, OR195041 and OR195040, respectively. A phylogenetic tree was constructed using the concatenated three gene sequences of JH-37 and that of the related Fusarium species based on Maximum Likelihood (Fig.2). JH-37 was grouped together with the F. armeniacum strain CBS 485.94 (AB587001, GQ915501, GQ915485), and shared 99.9 % concatenated sequence identity. The three gene sequences of the strain JH-37 shared 100%, 99.85%, 99.89% identity to F. armeniacum strain CBS 485.94 using MEGA 7 software (Kuma et al. 2016) analysis, and with 94%, 95% and 100% coverage by BLAST analysis. The colony of JH-37 on PDA at 25°C for 5 days was white with yellow-brown pigmentation in the center (Fig. 1B-C). From 10-day-old cultures grown on Spezieller Nahrstoffarmer agar (SNA), macroconidia (n = 50) were falcate, slender, curved dorsiventrally, tapering towards both ends, 3 to 4 septate, and measured 24.2 to 50.0 × 2.6 to 4.2 µm. The microconidia (n = 50) were straight or slightly curved, septate 0 to 2, and measured 6.8 to 20.0× 2.1 to 3.7 µm (Fig.1D-F). These morphological characteristics were consistent with Fusarium spp. (Leslie and Summerell 2006). A pathogenicity test of JH-37 was performed on potted lily ('Tresor') under greenhouse conditions. Healthy lily bulbs were selected and one bulb was sown in soil of each pot. Inoculation was performed 60 days after sowing. Bulbs of the lily plants were wounded with needles and inoculated with 5 mL of conidia suspension (1×107 conidia/mL) in the soil around bulb or an equal amount of sterilized water as a control. This experiment had three replicates. After 15 days of inoculation, typical symptoms of bulb rotten, and leaf wilt, similar to the original field symptoms, appeared on the inoculated plants but not on the controls (Fig.3). The same fungus was reisolated from the diseased plants, as identified based on morphology and molecular evidence, which confirmed the Koch's postulate. To our knowledge, this is the first report that F. armeniacum caused Fusarium wilt on Lilium spp. in China. Further, our result could help to develop effective disease management strategies against lily wilt disease.
RESUMO
Melon (Cucumis melo L.) is a member of the Cucurbitaceae family, and is an important economic and horticultural crop. In March 2022, melon plants in greenhouses exhibited severe leaf yellow spot symptoms in Changjiang County (109°13'N, 19°28'E), Hainan Province. The incidence of the disease was about 30-50%. Lesions initially appeared as yellow dots on leaves and expanded irregularly. Gradually, brown spots appeared, and finally the whole leaves turned yellow and resulted in blighting and death of foliage (Figure 1.). A total of four symptomatic plants were sampled from about 0.2 ha of an area. Symptomatic leaves were excised, surface disinfected with 2% (w/v) NaOCl, rinsed three times with sterile distilled water, and placed on potato dextrose agar (PDA) followed by incubation at 25°C in the dark for 5 days. The pure cultures were obtained by the hyphal-tip method. A total of eight fungal isolates with similar colony characteristics were recovered from the four symptomatic plants. Three DNA fragments (ITS, TEF1, and RPB2) of the eight isolates showed 100% sequence identity based on the molecular identification methods described below. Therefore, one of the isolates, M2JP-3, was chosen for identification and test of the pathogenicity. The colony of M2JP-3 on PDA at 25°C for 5 days was white with yellow-brown pigmentation in the center (Figure 2A-B). From 10-day-old cultures grown on CLA (Fisher et al. 1982), macroconidia (n = 50) were falcate, slender, curved dorsiventrally, tapering towards both ends, 3 to 7 septate, and measured 24.5 to 52.1 x 3.7 to 4.7 µm. The microconidia (n = 50) were straight or slightly curved, septate 0 to 2, and measured 9.9 to 16.3 x 2.5 to 3.7 µm (Figure 2C-E). For molecular identification, genomic DNA was extracted using the method previously described (Khan et al. 2021),the internal transcribed spacer (ITS), translation elongation factor 1α (TEF1) and DNA-dependent RNA polymerase subunit II (RPB2) were amplified, respectively, using primers ITS1/ITS4 (White et al. 1990), EF1/ EF2 (O'Donnell et al. 1998), and 5F2/7cR (Reeb et al. 2004). The 529 bp (ITS), 723 bp (TEF1), and 965bp (RPB2) sequences were deposited in GenBank with acce. nos. OP303211, OP312675 and OP312674, respectively. A phylogenetic tree was constructed using the concatenated three gene sequences of M2JP-3 and that of the Fusarium incarnatum-equiseti species complex (FIESC) (Xia et al. 2019) based on Maximum Likelihood (Figure 3). M2JP-3 was grouped together with the F. pernambucanum strain NRRL 32864 (accession no. GQ505702 for ITS, GQ505613 for TEF1and GQ505791 for RPB2), and shared 100% concatenated sequence identity. For pathogenicity tests of M2JP-3, seeds of melon cultivar Jinmeiren were surface disinfected and sowed in soil in three replicated pots in a greenhouse at 26 °C under natural light. Healthy leaves of the melon plants were wounded with needles and inoculated with mycelial plugs of M2JP-3 or PDA plugs as control. . Symptoms similar to the original greenhouse symptoms were observed at 7 days after inoculation (Figure 4). The control leaves were asymptomatic. The same fungus was reisolated from the inoculated leaves, as identified based on morphology and molecular evidence, which confirmed the Kochs' postulates. To our knowledge, this is the first time Fusarium pernambucanum has been recorded causing leaf yellow spot disease on melon. Further, findings of the present study will help to develop effective disease management strategies against Fusarium pernambucanum Leaf Yellow Spot on melon in China.
