RESUMO
Zizania latifolia is perennial plant, belonging to the rice tribe (Oryzeae) of the grass family Poaceae (Xu et al. 2020), which is also called jiaobai in China and commonly consumed as a vegetable crop. In 2022, a sheath rot occurred on Z. latifolia plants in Lishui, the Zhejiang Province of China. Symptoms occurred on the leaf sheath and initially showed as water-soaked chlorotic spots, later enlarging to irregular, elliptic, and elongated dark brown necrotic lesions. Later, lesions fused and extended to most of the leaf sheath leading to wilting. Almost 60% of the surveyed Z. latifolia plants in 100 hectare were affected. Diseased samples were collected for pathogen isolation. Symptomatic tissues were taken from the edge of lesions, sterilized for 10 s in 70% ethanol, then 2 min in 1% NaClO, washed three times with sterile distilled water, and placed on potato dextrose agar (PDA) at 26 °C in the dark. Fungal colonies displaying similar morphology were picked and purified by single spore isolation. In total, 8 isolates were obtained from 8 plant samples. When cultured on PDA, fungal colonies were white, gradually turning pale yellow with time. Macroconidia only were produced on Carnation leaf agar (CLA) and were hyaline, slender, falcate with single foot cells, 3 to 5 septate, and measured 29 to 50 µm × 3.75 to 5.0 µm. Chlamydospores were globose to subglobose and measured 6.8 to 16.5 µm. These morphological features were consistent with the description of Fusarium asiaticum (Leslie and Summerell 2006). For molecular identification, the partial translation elongation factor 1 alpha (TEF1-α) gene and RNA polymerase II second largest subunit (RPB2) gene of three representative isolates were amplified and sequenced (O'Donnell et al. 1998). These sequences were identical to each other, and one representative, Z-3-1, was deposited in GenBank (Accession No. OQ129437 and OQ858619, respectively). Analysis of the TEF1-α and RPB2 sequences of Z-3-1 showed that they were 99.85% (688/689) and 100% (945/945) identical to F. asiaticum strain Daya350-3 (KT380124) and MRC 1976 (MH582121), respectively, in NCBI, and had 99.38% and 100% identity to F. asiaticum strain CBS 110257 (AF212451 and JX171573) in Fusarium-ID. A combined phylogenetic tree based on the TEF1-α and RPB2 sequences showed that Z-3-1 was clustered with F. asiaticum using the neighbor-joining algorithm. Pathogenicity testing was conducted by inoculating potted Z. latifolia plants with a 1×105 conidial suspension of isolate Z-3-1, which was prepared by culturing the fungal strain in PDB at 26°C for 4 days in a shaker incubator. Conidial suspensions (1 mL) were dropped onto sheaths of potted Z. latifolia plants with sterile water serving as controls. All inoculated plants were covered with plastic bags and maintained in a humid growth chamber at 26°C with a photoperiod of 16 h. The inoculation experiment was repeated twice with 5 replicates per test. Four days later, the sheaths of potted inoculated plants displayed symptoms similar to those observed in the field. No symptoms were observed on control plants. Fusarium asiaticum was re-isolated specifically from the symptomatic inoculated Z. latifolia plants and confirmed by morphological and molecular methods, thus fulfilling Koch's postulates. Fusarium asiaticum has been reported to be a pathogen of other plants in China, such as Ligusticum (Zhu et al. 2022) and Setaria italica (Kong et al. 2022). To our knowledge, this is the first report of F. asiaticum causing sheath rot of Z. latifolia in China. The identification of the pathogen is the first step in developing appropriate field management strategies for this new disease.
RESUMO
Aloe vera (L.) Burm f., which belongs to the family Aloaceae, is a perennial succulent plant and cultivated for its medicinal, cosmetic, vegetable and ornamental uses. In summer of 2021, about 15% (60 infected among 400 surveyed plants) of A. vera (A. barbadensis) plants in two gardens in Lishui, Zhejiang Province of China showed symptoms of southern blight disease. Symptomatic plants primarily exhibited slightly sunken water-soaked, dark brown lesions on taproot and basal part of the stems. As the disease progressed, leaves in the basal part of stems and subsequently the whole plant rotted and withered, with white mycelial mats occurring on infected stems and leaves. Numerous brown, spherical sclerotia were observed on the colonized tissues and soil surfaces around the infected plants. Mycelial fragments and sclerotia from symptomatic leaves were plated directly to potato dextrose agar (PDA) amended with 100 µg/ml streptomycin and incubated at 26°C in the dark. By hyphal-tip method, a total of five pure isolates were obtained from five diseased leaf samples. When cultured on PDA at 26°C for three days, colonies showed white and thick aerial mycelium, with a radial growth rate of 23.7 mm/day. Typical clamp connection structures were observed microscopically after three days and numerous globoid, rapeseed shape sclerotia, measuring 1 to 2 mm in diameter (n=50) formed after six days. These sclerotia were initially white and gradually turned dark brown with age. On the basis of morphological characteristics, the fungal isolates were identified as Athelia rolfsii (Curzi) C.C. Tu & Kimbr (anamorph Sclerotium rolfsii Sacc) (Mordue 1974). The internal transcribed spacer (ITS) and translation elongation factor 1-α gene (TEF1) regions of a representative isolate LHBJ2-4 were amplified and sequenced using the primers ITS4/ITS5 (White et al. 1990) and EF1/EF2, respectively (accession no. MZ956758 and OL365370). BLASTn search showed that the amplified ITS and TEF1 sequences had 99.71% (680/682 bp) and 99.80% (498/499 bp) identity with the A. rolfsii isolates CBS 115.22 (MH854711.1) and Sr_286 (JF267815), respectively. Neighbor-joining phylogenetic tree based on the ITS sequences revealed that LHBJ2-4 clustered with A. rolfsii isolates. For pathogenicity test, three potted A. vera plants (~30 cm tall) were inoculated by placing a 0.5 cm mycelial plug of isolate LHBJ2-4 (three-day old) at the base of each A. vera plant. Three A. vera plants inoculated with sterile PDA plugs served as controls. All the inoculated plants were placed in a growth chamber at 27°C under a 12/12 h light/dark cycle. The inoculation assays were carried out twice. After 5 to 7 days, stem bases of the inoculated plants showed brown lesions that were similar to those observed in the field. However, control plants remained symptomless. Athelia rolfsii was re-isolated from all the inoculated plants and identified using morphological and molecular method described above, thus confirming Koch's postulates. Although A. rolfsii has been reported to cause disease on A. vera in India (Dubey and Pandey 2009), to the best of our knowledge, this is the first report of A. rolfsii causing southern blight on A. vera in China. Because A. rolfsii has a wide host range and is difficult to control (Punja 1985), occurrence of southern blight in China might be a serious threat for A. vera production and appropriate management strategies should be developed to control this disease.
RESUMO
Hemerocallis citrina Baroni, also called yellow flower vegetable (huang hua cai in Chinese), is belonging to the family Xanthorrhoeaceae and is widely planted in China, the Korea Peninsula and Japan for ornamental purposes and vegetable value. In addition, they could also be used as a traditional Chinese medicinal and modern medicinal plant (Du et al. 2014). In August 2019, a leaf spot disease was observed on H. citrina plants in Zhejiang Province of China, with approximately 85% incidence in almost 700 ha. Symptoms were firstly displayed as small, water-soaked, pale chlorotic spots, with yellow halos enlarged into large fusiform spots with brown edge and gray centers. Later, infected leaves were badly damaged and became wilted. Small pieces of infected tissue were excised from the margin of necrotic lesions, surface disinfected with 70% ethanol for 8s, 0.1% HgCl2 for 1 min, rinsed with sterile distilled water for three times, and incubated on potato dextrose agar (PDA, amended with 100 mg/L streptomycin sulfate) at 26°C in the dark. Fungal colonies with similar cultural morphology were consistently obtained from repeated isolations. When cultured on PDA, colonies were villose, regular, grayish-green, and turned gray-brown, with the reverse side became reddish-brown. Chlamydospores were gray, unicellular or multicellular, nearly spherical, 11 to 27 × 10 to 23 µm. Pycnidia and conidia were produced on PDA when the fungal colonies were exposed to ultraviolet light for 12 h with a distance of 40 cm to the late source. Pycnidia were brown, mostly spheroid, and measured 90 to 138 × 120 to 210 µm. Conidia were hyaline, ellipsoidal, unicellular, aseptate, 4.3 to 5.5 × 1.8 to 2.4 µm. These morphological characteristics agreed with the descriptions of Epicoccum sorghinum (Zhou et al. 2018). The DNA of a representative strain HHC6-2 was extracted using CTAB method and the rDNA internal transcribed spacer (ITS), actin (ACT) and ß-tubulin (TUB) genes were amplified and sequenced, using the primers ITS4/ITS5 (White et al. 1990), ACT512F/ACT783R (Carbone and Kohn 1999) and Bt-1/Bt-2 (Glass and Donaldson 1995), respectively. BLASTn searches of the resulting ITS, ACT and TUB sequences (accession nos. MW073403, MW080522, MW080521) revealed 98.58 to 100% identity to the E. sorghinum sequences (MT125854, MN956831 and MF987525). The pathogenicity test was carried out by inoculation of potted H. citrina plants using conidial suspensions. H. citrina seedlings were planted in pots with sterilized soil. Before inoculation, leaves were surface-disinfected with 70% ethanol and sterile distilled water. Leaves were inoculated by placing small droplets of conidial suspensions (105 conidia/ml) on one side of the midvein, and 3 to 5 drops were used per leaf. Sterile water was used as control. All the inoculated plants were placed in humid chambers at 25°C for 48h, and then maintained in a greenhouse at 25°C with a 16 h day-8 h night cycle. The pathogenicity assays were performed twice with three replications. Four days after inoculation, yellow to brown spots resembling those observed in the fields developed on the inoculated leaves. However, no symptoms were observed on the controls. E. sorghinum was re-isolated and identified based on morphological and molecular techniques as described above. To our knowledge, this is the first report of E. sorghinum causing leaf spot on H. citrina. It seems to be a threat for H. citrina planting in China and should be considered in order to reduce losses caused by this disease. This study might provide the basis for diagnosis and control of the disease.
RESUMO
Aloe vera (L.) Burm f. is a perennial herb belonging to the family liliaceae. It is widely grown for medicinal, cosmetic and vegetable use. In 2018 and 2019, a root rot disease occurred on potted A. vera plants in a nursery in the Hunan Province of China. Symptoms of the disease include water soaking lesions, brown spots on taproot or basal part of the stem. The plants were easy to pull out when the taproot is rotten or necrotic. As the disease progressed upward, leaves in the basal part of stems became red-brown and gradually fell off. In severe cases, the whole plants became rotten and wilted. For isolation purposes, diseased tissues were excised from the lesion margins, surface disinfested with 70% ethanol for 10 s, 0.1% HgCl2 for 2 min, rinsed with sterile water thrice, and then placed on potato dextrose agar (PDA) and incubated at 26°C for 3 days in the dark. When cultured on PDA, fungal strains with similar morphology were consistently isolated and purified by single spore isolation. Colonies showed thick, pink aerial mycelium with a growth rate of 1.3 cm /day. The pigmentation was more intense in the colony center and became pale orange and white at the edge of colony. When cultured on SNA (Spezieller Nährstoffarmer agar), the fungus showed less pigmentation and thinner hyphae. Microconidia were abundantly produced, clavate and oval to kidney shaped, 7.1 to 15.2 µm × 2.5 to 5.1 µm, with 0 to 1 transverse septa. Macroconidia were sickle shaped, slender, slightly incurved in apical cell and foot-shaped in the basal cell, measured 27.9 to 53.2 µm × 2.5 to 3.5 µm, with 3 to 5 septa. These morphological characteristics were similar with those of Fusarium spp. (Booth 1971). For molecular identification, genomic DNA of the fungus was extracted by cetyl trimethyl ammonium bromide method. A portion of EF-1α (translation elongation factor 1-α) and RPB1 (the largest subunit of RNA polymerase) genes were amplified and directly sequenced using the EF-1/EF-2 and Fa/G2R primers (O'Donnell et al. 2010). The EF-1α and RPB1 were deposited in the GenBank with accession numbers MT755386 and MT755387. The EF-1α and RPB1 had 97.14% (ID FD_01334) and 99.62% identity (FD_03853), respectively, to F. xylarioides strains in the Fusarium-ID database (Geiser et al. 2004). In addition, the EF1-a showed 96.825% identity to the F. lateritium CBS 119871(AM295281) (a synonym of F. xylarioides), and the RPB1 showed 99.623% identity to the F. xylarioides NRRL 25486 (JX171517.1). Accordingly, the fungus was putatively identified to be F. xylarioides. For pathogenicity assay, A.vera seedlings were pot planted using sterilized nursery soil and inoculated with conidia suspension (1 × 105 conidia/ml), which were eluted from 7-day-old PDA cultures with sterilized water, according to the method described previously (Vakalounakis et al. 2015). The collar of each potted plant was poured with 20 ml of conidia suspensions. Plants mock inoculated with sterile water were used as control. All the inoculated plants were placed in a growth chamber at 25°C under 12/12 h light/dark cycle. The inoculation assays were carried out twice, with each one had three replicated plants. After 30 days, rot symptoms seen from the roots and basal part of stems were observed on the inoculated plants, but no visible symptoms were observed on control plants. The fungus was re-isolated from the inoculated plants and identified to be F. xylarioides by morphological and molecular characteristics, thus confirming Koch's postulates. As we know, many Fusarium species have been reported to cause root and stem rot disease in A.vera such as the F. oxysporum (Ji et al. 2007) and F. solani (Vakalounakis et al. 2015). However, to the best of our knowledge, this is the first report of F. xylarioides causing root and stem rot disease of A.vera in China. The identification of the pathogen fungus might provide a foundation for taking appropriate control strategies to this disease.
