First Report of Fusarium solani Causing Leaf Sheath Rot of Bush lily in China

Bush lily (Clivia miniata) is an important indoor flower. It is the city flower of Changchun City and has important ornamental and medicinal value in China where it is culitvated on an area of 125 hectare. During the summer of 2018, symptoms of a leaf sheath rot disease were observed on bush lily in 103 greenhouses in Changchun city, Jilin Province. The disease incidence ranged from 25 to 60% in 11 surveyed greenhouses. At the early stage, the diseased plants displayed symptoms as initial leaf sheath lesions. Progressively, the whole leaves wilted, and even the plant ultimately died. Once a leaf exhibits leaf sheath lesions, the whole plant’s ornamental value significantly drops. To identify the pathogen, symptomatic leaves were cut into pieces, surface sterilized, placed on potato dextrose agar (PDA) and incubated for 7 days at 25°C in the dark (Cao et al. 2013; the e-Xtra description for details). Fusarium single-spore isolates were obtained from characteristic colonies (Leslie et al. 2006). Two single-spore isolates were selected for further study. The isolates were identified as Fusarium spp. based on microscopic morphology on PDA. Fusarium-like colonies were white to slightly yellow with abundant cottony mycelia. Single or two-celled (single septum) microconidia were reniform or oval, 8.0 to 9.6×4.0 to 6.0m in size. The elongated conidiophores bearing microconidia in monophialides were observed (Summerbell et al. 2002). Macroconidia were abundant, sickle shaped, 18.8 to 34.8×6.4 to 6.8m, with one to three septa (Taylor et al. 2019). For molecular identification, five regions of ITS, EF1-α, RPB1, RPB2 and β-tubulin genes were amplified and sequenced. Sequences of five different regions exhibited at least 97.98% similiarity with the corresponding DNA sequences in F. solani species complex (FSSC) (the e-Xtra description for details). The phylogenetic analysis based on the EF1-α, RPB1, RPB2 and β-tubulin region sequences revealed that the isolated strain in this study was clustered with only F. solani species in the phylogenetic tree for each region. Based on morphological and molecular analysis, the isolated fungal strains were identified as F. solani. Pathogenicity was confirmed by injecting a conidial suspension (106 spores/mL) of the isolated strains in to surface surface-disinfested leaf sheath of 2-year-old potted healthy plants. As a negative control, four plants were injected with sterilized water. All plants were kept in a greenhouse with controlled conditions: 26°C, 50% to 75% relative humidity. The similar rot symptoms were observed on the leaf sheathes in the inoculated plants 30 days after inoculation whereas the control plants remained asymptomatic. The fungi reisolated from the experimental plants were confirmed to be F. solani by morphology and sequences analysis, thus completing Koch’s postulates. To the best of our knowledge, this is the first report of F. solani causing leaf sheath rot of bush lily in China, where this pathogen has been reported to cause rot diseases of other economically important ornamental plants such as Phalaenopsis, Dendrobium according to the U.S. National Fungus Collections (Farr et al. 2020). In recent years, other Fusarium species have been reported to cause rot diseases on bush lily, including F. proliferatum and F. oxysporum (Farr et al. 2020). This study will also provide critical information on the causal agent for growers to implement disease management strategies.

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First Report on Ovate-leaf Atractylodes Damping-off Caused by Fusarium oxysporum species Complex in South Korea

Plant Disease ◽

10.1094/pdis-05-21-1107-pdn ◽

2021 ◽

Author(s):

Oliul Hassan ◽

Taehyun Chang

Keyword(s):

South Korea ◽

Fusarium Oxysporum ◽

Species Complex ◽

Disease Incidence ◽

Fusarium Species ◽

Morphological Characteristics ◽

Conidial Suspension ◽

Sterile Water ◽

Damping Off ◽

First Report

In South Korea, ovate-leaf atractylodes (OLA) (Atractylodes ovata) is cultivated for herbal medicine. During May to June 2019, a disease with damping off symptoms on OLA seedlings were observed at three farmer fields in Mungyeong, South Korea. Disease incidence was estimated as approximately 20% based on calculating the proportion of symptomatic seedlings in three randomly selected fields. Six randomly selected seedlings (two from each field) showing damping off symptoms were collected. Small pieces (1 cm2) were cut from infected roots, surface-sterilized (1 minute in 0.5% sodium hypochlorite), rinsed twice with sterile water, air-dried and then plated on potato dextrose agar (PDA, Difco, and Becton Dickinson). Hyphal tips were excised and transferred to fresh PDA. Six morphologically similar isolates were obtained from six samples. Seven-day-old colonies, incubated at 25 °C in the dark on PDA, were whitish with light purple mycelia on the upper side and white with light purple at the center on the reverse side. Macroconidia were 3–5 septate, curved, both ends were pointed, and were 19.8–36.62 × 3.3–4.7 µm (n= 30). Microconidia were cylindrical or ellipsoid and 5.5–11.6 × 2.5–3.8 µm (n=30). Chlamydospores were globose and 9.6 –16.3 × 9.4 – 15.0 µm (n=30). The morphological characteristics of present isolates were comparable with that of Fusarium species (Maryani et al. 2019). Genomic DNA was extracted from 4 days old cultures of each isolate of SRRM 4.2, SRRH3, and SRRH5, EF-1α and rpb2 region were amplified using EF792 + EF829, and RPB2-5f2 + RPB2-7cr primer sets, respectively (Carbone and Kohn, 1999; O'Donnell et al. 2010) and sequenced (GenBank accession number: LC569791- LC569793 and LC600806- LC600808). BLAST query against Fusarium loci sampled and multilocus sequence typing database revealed that 99–100% identity to corresponding sequences of the F. oxysporum species complex (strain NRRL 28395 and 26379). Maximum likelihood phylogenetic analysis with MEGA v. 6.0 using the concatenated sequencing data for EF-1α and rpb2 showed that the isolates belonged to F. oxysporum species complex. Each three healthy seedlings with similar sized (big flower sabju) were grown for 20 days in a plastic pot containing autoclaved peat soil was used for pathogenicity tests. Conidial suspensions (106 conidia mL−1) of 20 days old colonies per isolate (two isolates) were prepared in sterile water. Three pots per strain were inoculated either by pouring 50 ml of the conidial suspension or by the same quantity of sterile distilled water as control. After inoculation, all pots were incubated at 25 °C with a 16-hour light/8-hour dark cycle in a growth chamber. This experiment repeated twice. Inoculated seedlings were watered twice a week. Approximately 60% of the inoculated seedlings per strain wilted after 15 days of inoculation and control seedlings remained asymptomatic. Fusarium oxysporum was successfully isolated from infected seedling and identified based on morphology and EF-1α sequences data to confirm Koch’s postulates. Fusarium oxysporum is responsible for damping-off of many plant species, including larch, tomato, melon, bean, banana, cotton, chickpea, and Arabidopsis thaliana (Fourie et al. 2011; Hassan et al.2019). To the best of our knowledge, this is the first report on damping-off of ovate-leaf atractylodes caused by F. oxysporum in South Korea. This finding provides a basis for studying the epidemic and management of the disease.

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First Report of Walnut Canker Caused by Fusarium incarnatum from India

Plant Disease ◽

10.1094/pdis-04-11-0352 ◽

2011 ◽

Vol 95 (12) ◽

pp. 1587-1587

Author(s):

B. Singh ◽

C. S. Kalha ◽

V. K. Razdan ◽

V. S. Verma

Keyword(s):

Fusarium Species ◽

Juglans Regia ◽

Infected Plant ◽

Distilled Water ◽

Conidial Suspension ◽

State University ◽

Single Spore ◽

Jammu And Kashmir ◽

First Report ◽

Branch Dieback

While screening newly introduced cultivars of walnut (Juglans regia) at Bhaderwah (Mini Kashmir), Jammu and Kashmir, India in September 2008, 60% of grafted plants were found to be dying because of a cankerous growth observed on seedling stems. Later, these symptoms extended to lateral branches. In the surveyed nurseries, cvs. SKU 0002 and Opex Dachaubaria were severely affected by the disease. Cankers were also observed in all walnut nurseries in the area with several wild seedlings also being observed to be exhibiting similar cankerous symptoms on stem and branches. Necrotic lesions from cankerous tissues on seedling stems were surface disinfested with 0.4% NaOCl for 1 min and these disinfected cankerous tissues were grown on potato dextrose agar (potato-250 g, dextrose-15 g, agar-15 g, distilled water-1 liter). A Fusarium sp. was isolated consistently from these cankerous tissues, which was purified using single-spore culture. Carnation leaf agar was used for further culture identification (2,3). The fungal colony was floccose, powdery white to rosy in appearance when kept for 7 days at 25 ± 2°C. Macroconidia were straight to slightly curved, four to eight septate and 30 to 35 × 3.5 to 5.7 μm. These are characteristics consistent with Fusarium incarnatum (3). Pathogenicity was confirmed by spraying a conidial suspension (1 × 106 conidia/ml) onto bruised branches of 1-year-old walnut plants (cv. Opex Dachaubaria) while sterile distilled water sprays were used for the controls. Inoculated plants were incubated at 20 ± 2°C and 85% relative humidity for 48 h. Fifty days following inoculation, branch dieback followed by canker symptoms developed on inoculated plants. Control plants remained healthy with no symptoms of canker. F. incarnatum (Roberge) Sacc. was repeatedly isolated from inoculated walnut plants, thus satisfying Koch's postulates. Infected plant material has been deposited at Herbarium Crytogamae Indiae Orientalis (ITCC-6874-07), New Delhi. To our knowledge, this is the first report of walnut canker caused by F. incarnatum (Roberge) Sacc. from India. This fungus was previously reported to be affecting walnut in Italy (1) and Argentina (4). References: (1) A. Belisario et al. Informatore Agrario 21:51, 1999. (2) J. C. Gilman. A Manual of Soil Fungi. The Iowa State University Press, Ames, 1959. (3) P. E. Nelson et al. Fusarium Species. An Illustrated Manual for Identification. The Pennsylvania State University Press, University Park, 1983. (4) S. Seta et al. Plant Pathol. 53:248, 2004.

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First Report of Passalora Leaf Spot Caused by Passalora bougainvilleae on Bougainvillea in Mexico

Plant Disease ◽

10.1094/pdis-11-10-0787 ◽

2011 ◽

Vol 95 (6) ◽

pp. 775-775 ◽

Cited By ~ 1

Author(s):

V. Ayala-Escobar ◽

V. Santiago-Santiago ◽

A. Madariaga-Navarrete ◽

A. Castañeda-Vildozola ◽

C. Nava-Diaz

Keyword(s):

Uv Light ◽

Disease Incidence ◽

Morphological Characteristics ◽

The United States ◽

Pathogenicity Test ◽

Commonwealth Mycological Institute ◽

Conidial Suspension ◽

Single Spore ◽

First Report ◽

Bougainvillea Spectabilis

Bougainvillea (Bougainvillea spectabilis Willd) growing in 28 gardens during 2009 showed 100% disease incidence and 3 to 7% disease severity. Bougainvilleas with white flowers were the most affected. Symptoms consisted of light brown spots with dark brown margins visible on adaxial and abaxial sides of the leaves. Spots were circular, 2 to 7 mm in diameter, often surrounded by a chlorotic halo, and delimited by major leaf veins. Single-spore cultures were incubated at 24°C under near UV light for 7 days to obtain conidia. Pathogenicity was confirmed by spraying a conidial suspension (1 × 104 spores/ml) on leaves of potted bougainvillea plants (white, red, yellow, and purple flowers), incubating the plants in a dew chamber for 48 h and maintaining them in a greenhouse (20 to 24°C). Identical symptoms to those observed at the residential gardens appeared on inoculated plants after 45 to 60 days. The fungus was reisolated from inoculated plants that showed typical symptoms. No symptoms developed on control plants treated with sterile distilled water. The fungus produced distinct stromata that were dark brown, spherical to irregular, and 20 to 24 μm in diameter. Conidiophores were simple, born from the stromata, loose to dense fascicles, brown, straight to curved, not branched, zero to two septate, 14 × 2 μm, with two to four conspicuous and darkened scars. The conidia formed singly, were brown, broad, ellipsoid, obclavate, straight to curved with three to four septa, 40 × 4 μm, and finely verrucous with thick hilum at the end. Fungal DNA from the single-spore cultures was obtained using a commercial DNA Extraction Kit (Qiagen, Valencia, CA); ribosomal DNA was amplified with ITS5 and ITS4 primers and sequenced. The sequence was deposited at the National Center for Biotechnology Information Database (GenBank Accession Nos. HQ231216 and HQ231217). The symptoms (4), morphological characteristics (1,2,4), and pathogenicity test confirm the identity of the fungus as Passalora bougainvilleae (Muntañola) Castañeda & Braun (= Cercosporidium bougainvilleae Muntañola). This pathogen has been reported from Argentina, Brazil, Brunei, China, Cuba, El Salvador, India, Indonesia, Jamaica, Japan, Thailand, the United States, and Venezuela (3). To our knowledge, this is the first report of this disease on B. spectabilis Willd in Mexico. P. bougainvilleae may become an important disease of bougainvillea plants in tropical and subtropical areas of Mexico. References: (1) U. Braun and R. R. Castañeda. Cryptogam. Bot. 2/3:289, 1991. (2) M. B. Ellis. More Dematiaceous Hypomycetes. Commonwealth Mycological Institute, Kew, Surrey, UK, 1976. (3) C. Nakashima et al. Fungal Divers. 26:257, 2007. (4) K. L. Nechet and B. A. Halfeld-Vieira. Acta Amazonica 38:585, 2008.

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First report of Fusarium incarnatum causing fruit rot of litchi in China

Plant Disease ◽

10.1094/pdis-11-20-2393-pdn ◽

2021 ◽

Author(s):

Zhaoyin Gao ◽

Jiaobao Wang ◽

Zhengke Zhang ◽

Min Li ◽

Deqiang Gong ◽

...

Keyword(s):

Southern China ◽

Fusarium Species ◽

Morphological Characteristics ◽

Fruit Rot ◽

Conidial Suspension ◽

Single Spore ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Litchi Fruit ◽

His3 Gene

Litchi (Litchi chinensis Sonn.) is an indigenous tropical and subtropical fruit in Southern China with an attractive appearance, delicious taste, and good nutritional value (Jiang et al. 2003). In March 2020, brown rots were observed on nearly ripe litchi fruits (cv. Guihuaxiang) in an orchard of Lingshui county, Hainan province of China (18.615877° N, 109.948871° E). About 5% fruits were symptomatic in the field, and the disease caused postharvest losses during storage. The initial infected fruits had no obvious symptoms on the outer pericarp surfaces, but appeared irregular, brown to black-brown lesions in the inner pericarps around the pedicels. Then lesions expanded and became brown rots. Small tissues (4 mm × 4 mm) of fruit pericarps were cut from symptomatic fruits, surface-sterilized in 1% sodium hypochlorite for 3 min, rinsed in sterilized water three times, plated on potato dextrose agar (PDA) and incubated at 28℃ in the darkness. Morphologically similar colonies were isolated from 85% of 20 samples after 4 days of incubation. Ten isolates were purified using a single-spore isolation method. The isolates grown on PDA had abundant, fluffy, whitish to yellowish aerial mycelia, and the reverse side of the Petri dish was pale brown. Morphological characteristics of conidia were further determined on carnation leaf-piece agar (CLA) (Leslie et al. 2006). Macroconidia were straight to slightly curved, 3- to 5-septates with a foot-shaped basal cell, tapered at the apex, 2.70 to 4.43 µm × 18.63 to 37.58 µm (3.56 ± 0.36 × 28.68 ± 4.34 µm) (n = 100). Microconidia were fusoid to ovoid, 0- to 1-septate, 2.10 to 3.57 µm × 8.18 to 18.20 µm (2.88 ± 0.34 × 11.71 ± 1.97 µm) (n = 100). Chlamydospores on hyphae singly or in chains were globose, subglobose, or ellipsoidal. Based on cultural features and morphological characteristics, the fungus was identified as a Fusarium species (Leslie et al. 2006). To further confirm the pathogen, DNA was extracted from the 7-day-old aerial mycelia of three isolates (LZ-1, LZ-3, and LZ-5) following Chohan et al. (2019). The sequences of the internal transcribed spacer region of rDNA (ITS), translation elongation factor-1 alpha (tef1) gene, and histone H3 (his3) gene were partially amplified using primers ITS1/ITS4, EF1-728F/EF1-986R, and CYLH3F/CYLH3R, respectively (Funnell-Harris et al. 2017). The nucleotide sequences were deposited in GenBank (ITS: 515 bp, MW029882, 533 bp, MW092186, and 465 bp, MW092187; tef1: 292 bp, MW034437, 262 bp, MW159143, and 292 bp, MW159141; his3: 489 bp, MW034438, 477 bp, MW159142, and 474 bp, MW159140). The ITS, tef1, and his3 genes showed 99-100% similarity with the ITS (MH979697), tef1 (MH979698), and his3 (MH979696) genes, respectively of Fusarium incarnatum (TG0520) from muskmelon fruit. The phylogenetic analysis of the tef1 and his3 gene sequences showed that the three isolates clustered with F. incarnatum. Pathogenicity tests were conducted by spraying conidial suspension (1×106 conidia/ml) on wounded young fruits in the orchid. Negative controls were sprayed with sterilized water. Fruits were bagged with polythene bags for 24 hours and then unbagged for 10 days. Each treatment had 30 fruits. The inoculated fruits developed symptoms similar to those observed in the orchard and showed light brown lesions on the outer pericarp surfaces and irregular, brown to black-brown lesions in the inner pericarps, while the fruits of negative control remained symptomless. The same fungus was successfully recovered from symptomatic fruits, and thus, the test for the Koch’s postulates was completed. F. semitectum (synonym: F. incarnatum) (Saha et al. 2005), F. oxysporum (Bashar et al. 2012), and F. moniliforme (Rashid et al. 2015) have been previously reported as pathogens causing litchi fruit rots in India and Bangladesh. To our knowledge, this is the first report of Fusarium incarnatum causing litchi fruit rot in China.

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First Report of Leptosphaeria biglobosa Causing Black Leg on Raphanus sativus in Central China

Plant Disease ◽

10.1094/pdis-07-13-0728-pdn ◽

2014 ◽

Vol 98 (7) ◽

pp. 993-993 ◽

Cited By ~ 4

Author(s):

X. Cai ◽

L. Yang ◽

J. Zhang ◽

G. Q. Li

Keyword(s):

Raphanus Sativus ◽

Disease Incidence ◽

Central China ◽

Conidial Suspension ◽

Pcr Assay ◽

First Report ◽

Dna Fragment ◽

Plant Pathol ◽

Black Leg ◽

Β Tubulin

Chinese radish (Raphanus sativus) is an important vegetable grown widely in China. In 2010 to 2013, surveys for Leptosphaeria spp. on cruciferous vegetables were conducted in 17 counties in Hubei Province, China. Black leg symptoms on tuber roots and basal stems of radish were observed in Hanchuan, Jingmen, and Macheng counties. Disease incidence ranged from 2 to 25% in 10 surveyed radish fields. Five fungal isolates were obtained from diseased radish plants by surface-sterilizing radish tissue (5% NaOCl for 90 s, and then rinsed in sterilized water three times) and plating onto potato dextrose agar (PDA) plates incubated at 20°C. The isolations produced fluffy white colonies with a yellow pigment, and black-brown, globose pycnidia with pink conidial ooze formed after 10 days. Pycnidia were 150 to 200 × 80 to 100 μm. Conidia were hyaline, cylindrical, and 4 to 5 × 2 μm. The cultural and morphological characteristics of the isolates matched the description for Phoma lingam, anamorph of Leptosphaeria maculans and L. biglobosa (3). All five isolates were identified by PCR assay using the species-specific primers LbigF, LmacF, and LmacR (1), and isolate HCLB-1 was identified further by PCR cloning and analysis of the sequences coding for actin, β-tubulin, and the internal transcribed spacer (ITS) region of ribosomal DNA (3,4). Genomic DNA was extracted from mycelium of each isolate harvested from 7-day-old cultures in V8 broth using the CTAB method (5). A 444-bp DNA fragment was detected by PCR assay, suggesting that all five isolates belonged to L. biglobosa rather than L. maculans as the latter generates a 331-bp DNA fragment (1). The HCLB-1 sequences for ITS (587 bp, GenBank Accession. No. KC880981), actin (899 bp, KF307762), and β-tubulin (432 bp, KF220296) genes were 99 to 100% identical to those of L. biglobosa isolates in GenBank. All five isolates were tested for pathogenicity on R. sativus cultivars Duan Ye 13 and Qi Ye Hong. Cotyledons of 10-day-old radish seedlings and post-harvest mature roots were wounded using a sterilized needle, and 10 μl of a conidial suspension (1 × 107 conidia/ml) of each isolate was pipetted onto the wounded area on each cotyledon or root, respectively, with 12 cotyledons (= 24 wounded sites) and 1 root (= 6 wounded sites) inoculated/isolate. One wounded root and 12 wounded cotyledons inoculated with water were used as control treatments. Treated roots and seedlings were incubated at 20°C and 100% RH in the dark for 7 days, and under a 12 h light/12 h dark cycle for 12 days, respectively. While the control roots and cotyledons remained asymptomatic, the roots and cotyledons inoculated with all five test isolates formed black cankers and necrotic lesions, respectively, in the inoculated wounds. A fungus re-isolated from symptomatic roots and cotyledons resembled the original isolates in colony morphology and the 444-bp DNA fragment detected by PCR assay. No fungus was isolated from control seedlings or roots. Thus, L. biglobosa appears to be the causal agent of black leg observed on radish in Hubei, China. L. biglobosa was reported to infect wild radish (R. raphanistrum) (2). To our knowledge, this is the first report of L. biglobosa causing black leg on R. sativus. References: (1) S. Y. Liu et al. Plant Pathol. 55:401, 2006. (2) A. Maxwell and J. K. Scott. Australas. Plant Pathol. 37:523, 2008. (3) L. Vincenot et al. Phytopathology 98:321, 2008. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications, Academic Press, 1990. (5) J. Zhang et al. Mycologia 102:1114, 2010.

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First Report of Fusarium proliferatum Causing Garlic clove Rot in Russian Federation

Plant Disease ◽

10.1094/pdis-12-20-2743-pdn ◽

2021 ◽

Author(s):

Olga K Anisimova ◽

Timofey M Seredin ◽

Olga A Danilova ◽

Mikhail Filyushin

Keyword(s):

Rna Polymerase Ii ◽

Fruits And Vegetables ◽

Fusarium Species ◽

Taxonomic Status ◽

Basic Research ◽

Conidial Suspension ◽

Sterile Water ◽

Single Spore ◽

First Report ◽

Post Harvest

Garlic (Allium sativum L.) is a widely consumed bulbous crop both worldwide and in Russia. About 200,000 tons of garlic is produced in Russia annually (https://rosstat.gov.ru/). Significant pre- and post-harvest losses of garlic regularly occur due to Fusarium sp. (Taylor et al., 2013). Since September 2018, rotting has been observed in Russia during garlic bulb storage (data of the Federal Scientific Vegetable Center, FSVC, Moscow Region). The outer bulb surface looked healthy, but underneath the integumentary scales, the cloves had light brown and brown spots. When grown, diseased plants were characterized by root and bulb disruption and leaf drying; for some cultivars, up to 100% of plants died. In January 2020, cv. Strelets and Dubkovsky bulbs, collected in July 2019, with rot symptoms, were taken from the FSVC storage. Necrotic clove tissue fragments (0.2-0.5 cm) were cut, sanitized with 70% ethanol for 3 min, rinsed with sterile water, and incubated on potato dextrose agar (PDA) with 1 mg/ml ampicillin at 22°C in the dark. Four single-spore cultures were obtained from four diseased bulbs. After 6 days of incubation, the isolates produced abundant aerial white mycelia and acquired a purple pigmentation. The hyphae were hyaline with septation. All isolates (Dubkovsky, Dubkovsky 2, Strelets, and Strelets 2) produced numerous oval unicellular microconidia without septa, 4.1 to 11.6 × 1.3 to 3.4 µm (n = 50) and very few macroconidia with 3-4 septa (21 to 26 × 3 to 4 µm (n = 30)), narrowed at both ends. The cultural and conidial characteristics of the isolates corresponded to Fusarium species (Leslie and Summerell 2006). To determine the species, DNA was extracted from four isolates, and the internal transcribed spacer (ITS), and genes of translation elongation factor 1α (EF1α) and subunits 1 and 2 of DNA-directed RNA polymerase II (RPB1 and RPB2) were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), EF1/EF2 (O'Donnell et al. 1998a), RPB1-F5/RPB1-R8 (O’Donnell et al. 2010) and fRPB2-5F/fRPB2-7cR (Liu et al. 1999). The obtained sequences were identical for all four isolates. The isolate Strelets sequences were deposited in NCBI GenBank (MW149129 (ITS), MW161161 (EF1α), MW413302 (RPB1) and MW413303 (RPB2)); their analysis in MLST (http://fusarium.mycobank.org) showed 98.8-99.8% similarity to F. proliferatum (NRRL 13582, 13598 and others), which is part of the F. fujikuroi complex (O'Donnell et al. 1998b). The test on pathogenicity was performed two times according to (Leyronas et al. 2018). For this, three replicates of 10 cloves (cv. Strelets) were soaked in a conidial suspension (~106 conidia/ml; Strelets isolate) for 24 h. Ten control cloves were soaked in sterile water. The cloves were incubated on Petri dishes (5 cloves on a dish; on filter paper wettened with sterile water) in the dark at 23°C. After 5 days, brown lesions and white mycelium developed on the surface of the treated cloves. The taxonomic status of the fungus isolated from necrotic tissue was determined as F. proliferatum according to the ITS, EF1α, RPB1 and RPB2 analysis. Garlic basal and bulb rot is known to be caused by F. oxysporum f. sp. cepae and F. proliferatum (Snowdon 1990). This study is the first report of F. proliferatum causing rot of garlic bulbs during storage in Russia. F. proliferatum produces a variety of mycotoxins during bulb infestation, and our findings are important for diagnosing a Fusarium disease and the use of garlic crop in culinary and medicine. Funding The reported study was funded by Russian Foundation for Basic Research, project number 20-316-70009. References: Leslie, J. F., and Summerell, B. A. 2006. Page 224 in: The Fusarium Laboratory Manual. Blackwell, Oxford, UK. https://doi.org/10.1002/9780470278376 Leyronas, C., et al. 2018. Plant Dis. 102:2658 https://doi.org/10.1094/PDIS-06-18-0962-PDN Liu, Y.J. et al. 1999. Mol. Biol. Evol. 16: 1799 https://doi.org/10.1093/oxfordjournals.molbev.a026092 O'Donnell, K, et al. 1998a. Proc Natl Acad Sci USA. 95(5):2044. https://doi.org/10.1073/pnas.95.5.2044. O’Donnell, et al. 1998b. Mycologia 90:465 O’Donnell, K., et al. 2010. J. Clin. Microbiol., 48: 3708 https://doi.org/10.1128/JCM.00989-10 Snowdon, A. L. Pages 250–252 in: A Color Atlas of Post-Harvest Diseases and Disorders of Fruits and Vegetables. Vol. 1. 1990. Wolfe Scientific, London. Taylor, A, et al. 2013. Plant Pathol. 62:103. https://doi.org/10.1111/j.1365-3059.2012.02624.x White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.

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First Report of Leaf Spot Caused by Cladosporium tenuissimum on Panicle Hydrangea (Hydrangea paniculate) in China

Plant Disease ◽

10.1094/pdis-12-20-2640-pdn ◽

2021 ◽

Author(s):

Chenxu Li ◽

Peng Cao ◽

Chuanjiao Du ◽

Xi Xu ◽

Wensheng Xiang ◽

...

Keyword(s):

Dna Sequences ◽

Leaf Spot ◽

Disease Incidence ◽

Elongation Factor ◽

Flowering Plant ◽

Leaf Spot Disease ◽

Infected Leaf ◽

Conidial Suspension ◽

First Report ◽

Dark Green

Panicle Hydrangea (Hydrangea paniculate) is an ornamental flowering plant native to China and Japan. In August 2019, leaf spot symptoms with about 30% disease incidence were observed on panicle hydrangea in two grower fields (about 0.1 ha in total) of Northeast Agriculture University, China (126.72°E, 45.74°N). Symptoms initially appeared on the lower and older leaves and showed small subcircular brown spots with dark-brown edges on both sides. As the disease progressed, the necrotic spots enlarged, became irregular, coalesced, and the infected leaf blighted in approximately 2 weeks. Panicle hydrangea leaf samples (n=15) from different plants that showed spot symptoms were collected and surface sterilized with 70% ethanol for 10 s, followed by 0.5% NaClO treatment for 4 min, and rinsed in sterile water 3 times. Thereafter, leaf samples were placed on potato dextrose agar (PDA) and incubated at 25°C for 7 days. Fifteen hyphal-tipped pure cultures were obtained. Colonies growing on PDA for 7 days were olive green to dark green, exhibited a velvet-like texture and sometimes were radially furrowed and wrinkled. Margins varied from white gray to dark green without prominent exudates. The back of the plate showed dark green to black. Conidiophores were up to 180 to 600 µm long, 2.8 to 4.5 µm wide (n=50), subcylindrical-filiform, straight, septate, and unbranched or rarely branched. Ramoconidia were 0 to 1 septate, cylindrical to clavate, smooth-walled, 8 to 22 μm long (n=50). Conidia were single-celled, lemon-shaped, smooth-walled and 2.0 to 5.0 µm (diameter) (n=50). To confirm the identity, three genomic DNA regions, internal transcribed spacer (ITS), partial translation elongation factor-1 alpha (EF), and actin (ACT) of the representative isolate BAI-1 were amplified with primer pairs ITS1/4, EF1-728F/986R, and ACT-512F/783R, respectively (Bensch et al. 2012; Jo et al. 2018). DNA sequences of the isolate from ITS, EF, and ACT showed 99.81% (514/515 bp), 99.10% (219/221 bp), and 99.54% (216/217 bp) nucleotide identity with those of C. tenuissimum CBS 125995, respectively (GenBank accession nos. HM148197, HM148442, and HM148687). The sequences of isolate BAI-1 were deposited in GenBank (accession nos. MW045455, MW052465, and MW052466). To fulfill Koch’s postulates, five healthy 2-year-old panicle hydrangea plants grown in pots were surface sterilized with 70% ethanol, washed twice with sterile distilled water, and sprayed with a conidial suspension of strain BAI-1 (adjusted to 1×106 conidia/ml using a hemocytometer), maintained in a greenhouse at 25°C and 85% relative humidity. Five plants sprayed with sterilized water served as controls. The inoculated plants showed leaf spot symptoms that were similar to those previously observed in the fields after 7 days, whereas control leaves remained healthy. The fungus was reisolated from symptomatic leaves and its identity was confirmed by morphological and molecular method. These experiments were repeated twice. So far, C. tenuissimum was reported to cause leaf spot of alfalfa (Han et al. 2019) and castor (Liu et al. 2019). To our knowledge, this is the first report of leaf spot disease in panicle hydrangea caused by C. tenuissimum in China. Leaf spot has a negative effect on the aesthetic value of panicle hydrangea, and this report will assist with monitoring distribution of the disease as well as developing management recommendations.

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First report of Fusarium proliferatum causing root rot disease in Salvia miltiorrhizae in China

Plant Disease ◽

10.1094/pdis-09-20-1908-pdn ◽

2020 ◽

Author(s):

Jin Yang ◽

Fei Wang ◽

Yi Wen ◽

Suxia Gao ◽

Chuantao Lu ◽

...

Keyword(s):

Dna Sequences ◽

Root Rot ◽

Salvia Miltiorrhiza ◽

Disease Incidence ◽

Fusarium Proliferatum ◽

One Pot ◽

First Report ◽

Root Rot Disease ◽

Rot Disease ◽

Β Tubulin

Salvia miltiorrhiza Bunge is a herb plant used as a traditional Chinese medicine to cure cardiovascular disease. In December 2018, a root rot disease was observed on S. miltiorrhiza in four surveyed counties (Song, Yuzhou, Fangcheng, and Mianchi) in Henan province in China. The disease incidence ranged from 15 to 50% in 12 surveyed fields. At the early stage, the diseased plants were wilting with purple leaves. Leaves and branches became withered and fibrous roots became brown and rotted. The main roots of severely diseased plants also became rotted. The color of the stem surface turned from red to black, and the color of the stem xylem and phloem turned from dark red to brown. Eventually, the roots of diseased plants became completely rotted and the whole plants became dead, but no stink, which is different from Fusarium solani (Mart.) Sacc. (Yuan et al. 2015). Diseased root tissues (5×5×5 mm in size) were cut from diseased plants, surface-sterilized with 1% sodium hypochlorite for 1 min followed by dipping in 75% alcohol for 30 sec, rinsed in sterile distilled water for 3 times, air-dried on a sterilized filter paper in a laminar flow hood, placed on potato dextrose agar (PDA) containing 250 mg/l of streptomycin sulfate, and incubated at 28℃. Five isolates of Fusarium were obtained and purified using the single-spore isolation method. On PDA plates, the colonies were purple in color with formation of white aerial mycelia and reached 50 to 60 mm in diameter after incubation for 5 days. The colonies produced abundant microconidia on the colonies. The microconidia were 4.3 to 12.3 (10.0) × 2.1 to 3.5 (3.1) μm in size (n = 40), hyaline, ovoid or ellipse in shape. The conidiogenous cells were polyphialides. On mung bean media, the isolates formed macroconidia with 3 to 6 septae, fusiform in shape, slightly curved, 21.8 to 32.7 (31.4) × 2.6 to 4.3 (3.4) μm in size (n = 50). The morphological features of the five isolates were consistent with the description for Fusarium proliferatum (Matsush.) Nirenberg ex Gerlach & Nirenberg (Leslie and Summerell 2006). To further define the identity of the five isolates, molecular phylogenetic analysis was performed. The genomic DNA was extracted from all five isolates using the cetyl trimethylammonium bromide (CTAB) method. Five genes [nuclear ribosomal internal transcribed spacer (ITS) region, translation elongation factor 1-α (EF1α), β-tubulin gene, partial sequence for calmodulin (PRO), and RNA-dependent DNA polymerase II subunit (RPB2)] in F. proliferatum were amplified using primers pairs ITS1/ITS4, EF1T/2T, β-tubulin 2a/b, PRO1/2, and RPB2F/R, respectively (Glass and Donaldson 1995; Liu et al. 1999; Mulè 2004; O'Donnell et al. 1998; O'Donnell et al., 2010). The sequences (GenBank accession numbers: MT371373, MT371384, MT925651, MT925652, and MT934441, respectively) showed 99.6 to 100% identities to the corresponding DNA sequences in F. proliferatum (GenBank Acc. Nos. MK243486, MN245720, KJ12896, MN245721, and MK144327, respectively). All five isolates were tested for pathogenicity to fulfill the Koch's postulates. The 45-day-old healthy plants of S. miltiorrhiza grown in sterilized soil in pots (20 cm in diameter), one plant in one pot, were inoculated with conidial suspensions (1.0 × 107 cfu/ml) by pouring 10 ml conidial suspensions around the stem base in one pot. For each isolate, four plants were inoculated. Four plants were treated with sterilized water in the same volume as a control. The tested plants were placed in a growth room at 25°C (RH > 60%) with a 12 h photoperiod of fluorescent light. The pathogenicity assay was repeated for three times. The similar wilt symptoms were observed on the roots in the inoculated plants 30 days after inoculation but were not observed in the control plants. F. proliferatum was re-isolated from the infected roots, and its identity was confirmed by PCR with the primers described above. To our knowledge, this is the first report of F. proliferatum casing root rot disease on S. miltiorrhiza in China.

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First Report of Maize Stalk Rot Caused by Fusarium nelsonii in China

Plant Disease ◽

10.1094/pdis-01-21-0089-pdn ◽

2021 ◽

Author(s):

Xiaojie Zhang ◽

Cheng Guo ◽

Chunming Wang ◽

Tianwang Zhou

Keyword(s):

Fusarium Species ◽

Morphological Characteristics ◽

Molecular Characteristics ◽

Conidial Suspension ◽

Basal Cells ◽

Sterile Water ◽

Single Spore ◽

First Report ◽

Stalk Rot ◽

Maize Plants

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. References: Ahmad, A., et al. 2020. Plant disease.1542 https://doi.org/10.1094/PDIS-11-19-2511-PDN Astoreca, A. L., et al. 2019. Eur. J. Plant Pathol. 155:381. Lincy, S. V., et al. 2011. World J. Microbiol. Biotechnol. 27:981. Marasas, W. F. O., et al. 1998. Mycologia 90:505. Zhang, Y., et al. 2016. PLoS Pathog. 12:e1005485. Funding: This research was financially supported by National R & D Plan of China (No.2019QZKK0303); Ningxia Agriculture and Forestry Academy Science and Technology Cooperation Project (DW-X-2018019)

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First Report of Fusarium equiseti Causing Seedling Death on Sugar Beet in Minnesota, USA

Plant Disease ◽

10.1094/pdis-10-20-2102-pdn ◽

2021 ◽

Author(s):

Mohamed Fizal Khan ◽

Yangxi Liu ◽

Md. Ziaur Rahman Bhuyian ◽

Dilip Lashman ◽

Zhaohui Liu ◽

...

Keyword(s):

Sugar Beet ◽

Fusarium Species ◽

Morphological Characters ◽

Root Tip ◽

Economic Damage ◽

Fluorescent Light ◽

Conidial Suspension ◽

Single Spore ◽

Fusarium Equiseti ◽

First Report

In May 2019, sugar beet (Beta vulgaris L.) seedlings with symptoms of wilting and root tip discoloration and necrosis were found in Moorhead (46.5507° N, 96.4208° W), Minnesota, USA. Roots of infected seedlings were surface sterilized with 10% bleach for 15 seconds, rinsed with sterile distilled water and cultured on water agar (MA Mooragar®, Inc, CA) for 3 days at 23 ± 2°C. Isolates were transferred to carnation leaf agar (CLA) and incubated at room temperature (22°C) under fluorescent light for 14 days. Abundant macroconidia were produced in sporodochia. Macroconidia were 5- to 7-septate, slightly curved at the apex, and ranged from 35 to 110 ×1.2 to 3.8 μm. No microconidia were produced. Chlamydospores with thick, roughened walls were observed in chains or in clumps, and were ellipsoidal or subglobose. Single spore was transferred from CLA to potato dextrose agar (HIMEDIA Laboratories, India) produced abundant white mycelium and was pale brown where the colony was in contact with the media. The morphological features of the isolates were consistent with Fusarium equiseti (Corda) Sacc. (Leslie and Summerell 2006, Li et al. 2015). Genomic DNAs (NORGEN BIOTEK CORP, Fungi DNA Isolation Kit #26200) of two representative isolates were used for polymerase chain reaction (PCR). The second largest subunit of RNA polymerase (RPB2) was amplified by PCR with primers 5f2/7cr (O’Donnell et al. 2010). The amplified PCR product was sequenced and deposited in GenBank (accession number MW048778). A BLAST search in Genbank and the Fusarium MLST database showed 100% sequence alignment to F. equiseti with accession MK077037.1 and NRRL 25795, respectively. Pathogenicity testing was done using three sugar beet seedlings (Hilleshög proprietary material, Hilleshög Seed, LLC, Halsey, OR 97348) at cotyledonary stage grown in a pot (4˝×4˝×6˝) with six replicates. Seedlings were inoculated with F. equiseti conidial suspension (104 conidia ml-1 for 8 minutes) by the root dip method (Hanson, 2006). Mock inoculated plants were dipped in sterile water. Inoculated and control plants were placed in the greenhouse at 25 ± 2°C, and 75 to 85% relative humidity. One week later, inoculated seedlings showed root tip tissue discoloration similar to those observed in the field and non-inoculated seedlings were symptomless. This study was repeated. The fungus was re-isolated from diseased roots and confirmed to be F. equiseti based on morphological characters. Fusarium equiseti was reported on freshly harvested and stored beet in Europe but was not found to be pathogenic (Christ et al. 2011). Strausbaugh and Gillen (2009) reported the association of F. equiseti and root rot of sugar beet but did not report pathogenicity. This pathogen is reported in several crops including edible beans that is grown in rotation with sugar beet in several production areas (Jacobs et al. 2018). The most important Fusarium species reported to cause significant economic damage to sugar beet include F. oxysporum and F. secorum (Secor et al. 2014, Webb et. al. 2012). The presence of another pathogenic Fusarium species in sugar beet will require monitoring to determine how widespread it is and whether current commercial cultivars are resistant. To our knowledge, this is the first report of F. equiseti causing disease on sugar beet seedlings in Minnesota, USA.

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