• March 4, 2024

Duohua huangjing (Polygonatum cyrtonema Hua) seedling basal rot caused by Fusarium redolens in China

Duohua huangjing (Polygonatum cyrtonema Hua) seedling basal stem rot caused by Fusarium redolens in China Tao Tang1, Fanfan Wang1, Jie Guo1, Xiaoliang Guo1, Yuanyuan Duan1,Jingmao You1* 1 Institute of Chinese Herbal Medicines, Hubei Academy of Agricultural Sciences, Enshi, 445000, China. Duohua huangjing (Polygonatum cyrtonema Hua), a herbal medicine, that is mostly planted in several provinces in China. In April 2020, severe diseases with about 40% seedling losse was found in the Huangjing seedling base in Shiyan city, Hubei province. The symptoms included softening and decay of the roots and stem bases, a progressive yellowing and wilting of leaves, and finally being completely rotted. Small pieces of symptomatic stems (0.5 cm in length) and leaves (0.5 × 0.5 cm in size) were surface sterilized with 75% ethanol for 30 s, followed by 0.1% HgCl2 for 1 min, rinsed three times with sterile water, and then dried with sterilized absorbent paper.
The sections were placed on potato dextrose agar (PDA) medium containing 10 µg/ml of ampicillin and incubated at 25°C in the dark. After 3 days incubation, eight isolates with the same colony morphology were sub-cultured and purified by hyphal tip isolation. Macroconidia were sickle-shaped, 15.8 – 32.3 × 3.1 – 5.6 μm (n = 25), and three to five septate.
Microconidia were oval or kidney-shaped, 5.2 – 11.4 × 2.0 – 3.2 μm (n = 25), and zero to one septate. To confirm the identity of the pathogen, molecular identification was performed with strain HJCD1. Following DNA extraction, PCR was performed using the TSINGKE 2×T5 Direct PCR Mix kit. Target areas of amplification were the internal transcribed spacer (ITS) and translation elongation factor 1α (TEF-1α) using ITS1/4 (White et al. 1990) , EF1/EF2 (Taylor et al. 2016), respectively.
Following BLAST searches and phylogenetic reconstruction, the ITS region (GenBank MW485770.1) showed 99% identity with those of Fusarium redolens in GenBank (KU350713.1) and the TEF-1α (GenBank MW503930.1) showed 100% identity with F. redolens GenBank (MK922537.1). Pathogenicity tests were performed to fulfill Koch’s postulates. Huangjing seedlings were rinsed with sterile water, wiped clean with sterile absorbent paper, and transferred to a tray covered with wet filter paper to maintain high humidity.
The mycelial piugs of F. redolens HJCD1 were inoculated onto the surface of leaves and basal stems. Controls were inoculated with sterile PDA plugs. The inoculated seedlings were sealed with plastic wrap, and then cultivated in a 25 ℃ growth chamber with 16 h of light per day. The pathogen-inoculated plants Gentaur PCR Filter Tips exhibited etiolation and typical wilt symptoms after 4 days, whereas no symptoms were observed in the control plants. F. redolens was reisolated from the infected tissues, and colony morphology and ITS sequence of re-isolates were same as that of HJCD1. The pathogen has been reported previously in american ginseng in China (Fan et al. 2021), lentil in Pakistan (Rafique et al. 2020), and wild rocket in United Kingdom (Taylor et al. 2019).
However, to the best of our knowledge, this is the first report of F. redolent causing seelding basal rot on Duohua huangjing in China. References: White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Taylor, A., et al. 2016. Mol. Plant Pathol. 17:1032. https://doi.org/10.1111/mpp.12346 Fan, S. H., et al. 2021. Plant Dis. https://doi.org/10.1094/PDIS-11-19-2519-PDN Rafique, K., et al. 2020. Plant Dis. 9:104. https://doi.org/10.1094/PDIS-11-19-2519-PDN Taylor, A., et al. 2019. Plant Dis.6:103.
PDN Funding: Science Funds for Young Scholar of Hubei Academy of Agricultural Science (grant no. 2020NKYJJ20), National Modern Agricultural Industrial Technology System (grant no. CARS-21), Technology R&D Program of Enshi (grant no. D20190015), Science Funds for Young Scholar of Institute of Chinese Herbal Medicines, Hubei Academy of Agricultural Sciences (grant no. 2019ZYCJJ03), Key Laboratory of Integrated Management of Crops of Central China, Ministry of Agriculture, P. R. China / Hubei Key Laboratory of Crop Disease, Insect Pests and Weeds Control (grant no.2020ZTSJJ6).

First report of Rhizoctonia solani AG-4 HGI Causing Stem Canker on Fagopyrum tataricum (Tartary buckwheat) in China

Tartary buckwheat (Fagopyrum tataricum) is an ideal functional food source, which is well known to be gluten-free and rich in proteins, fats, vitamins, minerals, and flavonoids of various pharmaceutical uses, such as rutin, quercitin and epicatechin (Zhou, M et al. 2018). The Rhizoctonia solani AG-4 HGIII causing severe canker disease was first isolated from common buckwheat (F. esculentum) in Inner Mongolia of China (Zhou, H et al. 2015).
In 2018, sunken lesion and dark brown symptoms were observed on the root and stem of ten days old Tartary buckwheat in Liangshan (28°21’N, 103°19”E), Sichuan Province and Fenghuang (28°19′ N, 109°48′ E), Hunan Province in China. In the beginning, water-soaked spots appeared on the stem base, where gradually became rotten and necrotic, finally resulting in the damping-off and death of buckwheat seedlings.
This disease had over 40% incidence and lead to serious losses to the buckwheat production in 2018. To isolate the pathogens on Tartary buckwheat, ten plants with typical symptoms were collected from each location. The infected tissue was taken and cut into 3mm pieces from the margin between healthy and diseased tissue, surface sterilized with 1% sodium hypochlorite solution for 4 min, washed three times with sterile distilled water, dried on sterilized filter paper and then placed on potato dextrose agar (PDA) with 100 mg/ml streptomycin sulfate. After incubation at 28℃ in the dark for 2 days, mycelial tips of four fungal cultures were transferred to PDA plates for purification. Initially, colonies were pale white, and then turned brown after 2 days incubation.
The mycelium was hairy and concentrically whorled in the culture medium. Microscopic observation showed that the hyphae characteristically branched at right angles and had constriction at the base of hyphal branches. Nuclear staining showed that the hyphae cells were multinuclear. These morphological features revealed that the isolates belonged to R. solani (Sneh et al. 1991). Subsequently, the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region of one isolate was amplified by PCR (White et al. 1990) and sequenced (GeneBank accession no. MT078642) by Shanghai Majorbio Bio-pharm Technology Co.,Ltd. DNA was extracted by Fungal genomic DNA Extraction Kit, D3390-02, OMEGA. The BLAST similarity analysis showed a 99.96% match with R. solani AG-4 HGI (GenBank accession no. JQ343830) and 99.85% identity to R. solani AG-4 HGI isolate SX-8 (GenBank accession no. KJ170346) (Suli, Sun et al. 2015). Furthermore, the phylogenetic analysis performed by the neighbour-joining method (MEGA 7 software) showed that the isolate was clearly clustered with the group of R. solani AG-4 HGI (Ireland et al. 2015). Pathogenicity was tested in the greenhouse condition to satisfy Koch’s postulates. Tartary buckwheat plants of seven days old and fifty days old were respectively inoculated near the base of the stem neck with one mycelial plug contacted directly.
Ten plants in a pot were inoculated as one treatment, four pots were used for replicates. Control plants were inoculated with PDA medium plugs without fungi. All the plants were kept at 26℃ with 14 h light, 10 h dark and 96% humidity.
After five days (Suli, Sun et al. 2015), over 90% of the inoculated plants exhibited necrotic brown lesions on stems that was similar to those symptoms observed in the field, whereas control plants remained asymptomatic. The visible characteristics and ITS sequence of the pathogen re-isolated from symptomatic plants were in accordance with the original isolate (R. solani AG-4 HGI).
Based on disease symptoms in the fields, morphological characteristics, ITS sequence analysis, and pathogenicity assay, we concluded that R. solani AG-4 HGI was the principle cause of Tartary buckwheat blight in Liangshan, Sichuan Province and Fenghuang, Hunan Province in China. Previously, R. solani AG-4 HGI has been identified as a Chinese chive pathogen (Shi, Y et al. 2017). To the best of our knowledge, this is the first report of the natural occurrence of Rhizoctonia solani AG-4 HGI affecting Tartary Buckwheat in China. This finding is helpful for the early diagnosis and identification of the disease, which will be the guiding of effective control methods to the devastating disease at the early stage.

Phytophthora cactorum as a Pathogen Associated with Root Rot on Alfalfa (Medicago sativa) in China

Alfalfa (Medicago sativa) is the largest grown pasture crop in China due to its economic and ecological importance. During the summer season from June to August in 2018, stunted plants was frequently observed in alfalfa fields that have been established for two years in Jinchang, Gansu Province. The disease incidence of root rot ranged from 40% to 50%. Taproots of stunted plants showed red-brown to dark brown discolorations, and lateral roots were poorly developed. Shoots wilted with rotted taproots and lateral roots in severely affected plants.
Twenty symptomatic plants were collected and transported to the laboratory for pathogen isolations. Roots were washed under running tap water, cut into 2 to 3 mm pieces (40 pieces each plant), and then sterilized in 75% ethanol for 2 mins followed by three times washing with autoclaved distilled water. Surface dried pieces on autoclaved filter papers were put onto water agar and also a Phytophthora selective medium P5ARP(H) (Jeffers and Martin 1986). The plates were incubated at 22°C for 3 to 5 days and then the growing hypha were subcultured onto potato dextrose agar (PDA).
Thirty-two Phytophthora-like isolates were obtained and showed similar morphologies on PDA. Five isolates picked randomly were purified by single-hyphal-tip and plugs (4 to 5 mm) from PDA cultures were incubated in petri dishes with autoclaved distilled water at 22°C for 5 days. Sporangia, chlamydospores and oospores were examined. Sporangia were usually ovoid and sometimes appeared ellipsoid, with the length of 30.5-39.1 μm and width of 23.4-27.8 μm.
The diameter of chlamydospores was 29.6 to 42.5μm. Oospores had a diameter of 23.6 to 30.2 μm. The isolates were tentatively identified as P. cactorum based on these morphology characteristics (Montealegre et al. 2016). DNA of these isolates were extracted and PCR amplifications of the rDNA ITS region and cytochrome oxidase subunit I (Cox I) (Kroon et al. 2004) were conducted. Sequences of these isolates were then compared with reference sequences in GenBank using BLAST search.
The 866-bp ITS sequences had a sequence identity of 99% to 100% with P. cactorum (e.g. accession nos. EU662221, KJ128036). In addition, the 663-bp CoxI sequences showed 100% sequence identity with three P. cactorum isolates (accession nos. AB688156, HQ708234, EU660851). The ITS and CoxI sequences of one representative isolate Phy.c2 have been deposited in GenBank with the accession no. MT280033 and MT344138, respectively.
Pathogenicity of the five isolates (Phy.c1-Phy.c5) were determined on two-week-old alfalfa seedlings (cv. Longdong) grown from seeds. Inoculums were prepared by subculturing agar plugs from edges of PDA cultures into the flask with autoclaved millet seeds, and incubated at 22°C in darkness for two weeks and shaken by hand every two days to ensure uniform colonization. Seedlings were transplanted into pots (12 cm x 12 cm) filled with autoclaved potting mix infested with millet-seed inoculum of each isolate at a rate of 0.5% (w/w). Control seedlings for comparison were transplanted into pots with uninfested potting mix.
There were five seedlings per pot and twelve replicate pots for both inoculated and noninoculated treatments, and pots were kept under controlled environment room (22°C, 12 h photoperiod and 65% relative humidity) that were watered every two days to free draining. 87%~92% of the inoculated plants showed stunted symptoms with poorly developed and brown-discoloured roots three weeks after inoculation while the control plants were healthy with no root disease symptoms.
To fulfill Koch’s postulates, re-isolated cultures from discoloured root tissues were confirmed as the inoculated isolates by morphological examination and ITS sequencing. The five-purified isolates were submitted to the Grassland Culture Collection Center, Lanzhou University, with the accession nos. LZU-MsR-Phy.c1-Phy.c5. To our knowledge, this is the first report of P. cactorum as a pathogen of root rot on alfalfa in China.
Phytophthora spp. has been reported causing root rot on alfalfa in America, Australia and Canada, and other legumes such as chickpea, and many other crops worldwide (Musial et al. 2005; Tan and Tan 1986; Vandemark and Barker 2003), and P. cactorum was reported as a root rot pathogen on lavender in China (Chen et al. 2017). P. cactorum may be a significant pathogen associated with root rot in major commercial alfalfa-producing areas in China where are based on flood-irrigation during the growth season.

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