First Report of Fusarium Root Rot of Tobacco Caused by Fusarium sinensis in Henan Province China

Tobacco (Nicotiana tabacum L.) is an economically important crop in China, with an estimated production of 2.2 million tons every year. In June 2018, tobacco plants within the municipality of Sanmenxia (Henan, China) showed symptoms of wilting with leaf yellowing and stunting. Diseased plants exhibited severe necrosis that advanced through the main root (Figure 1 A). The symptoms were observed in nineteen surveyed tobacco fields, 60 ha in total, and approximately 25% of the plants were symptomatic. The disease resulted in a severe loss in tobacco leaf production. Five symptomatic tobacco plants were sampled. Diseased tissues from roots were surface sterilized in 75% ethanol and placed on potato dextrose agar (PDA) medium. Eighteen of the 25 diseased tissues had cultures growing from them, and all the cultures were white colonies with abundant aerial mycelium produced scarlet pigmentation on PDA. One pure culture was obtained by single-spore culturing (SL1). A 10-day-old culture grown on CLA (carnation leaf agar) produced macroconidia that were falcate, straight or slightly curved, 3-septate, 25-35×3.5-4.5 μm (average 26.8×3.7 μm) (n=50). Two types of microconidia (napiform and fusiform) were formed on CLA that were hyaline, with one to two cells. Napiform conidia were 4.5-9.3×3.8-5.9 (average 7.3×5.0 μm) (n=50); fusiform conidia were 6.9-15.8×1.8-3.1 (average 9.9×2.5 μm). Spherical chlamydospores (7-12.5 μm) (n=50) were terminal or intercalary and produced in clumps or in chains (Figure1 B-D). Morphological characteristics of the isolate were similar to the features of Fusarium sinensis previously described by Zhao and Lu (2008). Molecular identification was performed using partial sequences of EF1-α gene (primers EF1/EF2, O’Donnell et al. 1998). Maximum parsimony and maximum likelihood-based methods were fitted using MEGA 7 (Moreira et al. 2019，Figure 2). The isolate was also sequenced for β-tubulin (primers T1/Bt-2b, O’Donnell & Cigelnik 1997)，ribosomal RNA gene (LSU, LROR/LR5 primers, Vu et al. 2019) and rDNA-ITS (ITS 1/ ITS 4 primers, White et al. 1990). Sequences were deposited in GenBank under accession numbers MT947797 (EF1-α), MW484999 (β-tubulin), MW486649 (LSU) and MT907471 (ITS). The obtained EF1-α sequence was 98.10% identity with those of F. sinensis (MG670388.1) in the GenBank database, whereas the β-tubulin, LSU and ITS sequences showed 100% identities to the corresponding DNA sequences in F. sinensis (GenBank Acc. Nos. KX880370.1, NG_067454.1 and MH863232.1, respectively). Morphological and molecular results confirmed this species as F. sinensis (Zhao and Lu 2008). Pathogenicity tests were performed on tobacco seedlings grown on an autoclaved matrix (YC/T310-2009). Healthy 6-leaf stage tobacco seedlings were inoculated by pouring a 20 mL conidial suspension (1×106 conidia/mL-1) around the stem base of each plant, 30 plant were inoculated. Thirty control seedlings received sterilized water. All treatments were maintained for 30 days under greenhouse conditions with a 12-h light/dark photoperiod at 25±0.5℃ and 70% relative humidity. The assay was conducted three times. Root rot and foliage chlorosis similar to the ones observed on infected plants in the field were observed on the inoculated tobacco seedlings, whereas the control seedlings remained asymptomatic after 30 days (Figure1 E). The pathogen isolated from the inoculated plant exhibited morphological characteristics identical to F. sinensis and was identified by a partial EF1-α gene sequence. This disease has previously been reported as the causal agent of root and crown rot of wheat in China (Zhao and Lu 2008; Xu et al. 2018). To our knowledge, this is the first report of F. sinensis causing root rot on tobacco in China. Funding: Funding was provided by the Science and Technology Project of Henan Provincial Tobacco Company (2020410000270012), Independent Innovation Project of Hennan Academy of Agricultural Sciences (2020ZC18) and Research and Development project of Henan Academy of Agricultural Sciences (2020CY010). References: Moreira, G.M., et al. 2019 Plant Dis. O’Donnell, K., et al. 1998. Proc. Natl. Acad. Sci. USA 95:2011. O'Donnell, K., et al. 2008. J. Clin. Microbiol. 46:2477. Xu, F., et al. 2018. Front Microbiol. 9:1054. Zhao, Z.H., and Lu, G. Z., 2008. Mycologia, 100:746. The author(s) declare no conflict of interest. Keywords: tobacco root rot, Henan Province, Fusarium sinensis

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First Report of Stem Blight Caused by Calonectria colhounii (Anamorph Cylindrocladium colhounii) on Greenhouse-Grown Blueberries in the United States

Plant Disease ◽

10.1094/pdis-02-11-0117 ◽

2011 ◽

Vol 95 (9) ◽

pp. 1187-1187

Author(s):

J. J. Sadowsky ◽

T. D. Miles ◽

A. M. C. Schilder

Keyword(s):

United States ◽

North Carolina ◽

Root Rot ◽

The United States ◽

Vaccinium Corymbosum ◽

Conidial Suspension ◽

Centraalbureau Voor ◽

First Report ◽

Stem Lesions ◽

Β Tubulin

Necrotic stems and leaves were observed on 2- to 4-month-old, rooted microshoot plants (Vaccinium corymbosum L. ‘Liberty’ and ‘Bluecrop’, V. angustifolium Aiton ‘Putte’, and V. corymbosum × V. angustifolium ‘Polaris’) in a Michigan greenhouse in 2008 and 2009. As the disease progressed, leaves fell off and 80 to 100% of the plants died in some cases. Root rot symptoms were also observed. A fungus was isolated from stem lesions. On potato dextrose agar (PDA), cultures first appeared light tan to orange, then rusty brown and zonate with irregular margins. Chains of orange-brown chlamydospores were abundant in the medium. Macroconidiophores were penicillately branched and had a stipe extension of 220 to 275 × 2.5 μm with a narrowly clavate vesicle, 3 to 4 μm wide at the tip. Conidia were hyaline and cylindrical with rounded ends, (1-)3-septate, 48 to 73 × 5 to 7 (average 60 × 5.5) μm and were held together in parallel clusters. Perithecia were globose to subglobose, yellow, 290 to 320 μm high, and 255 to 295 μm in diameter. Ascospores were hyaline, 2- to 3-septate, guttulate, fusoid with rounded ends, slightly curved, and 30 to 88 × 5 to 7.5 (average 57 × 5.3) μm. On the basis of morphology, the fungus was identified as Calonectria colhounii Peerally (anamorph Cylindrocladium colhounii Peerally) (1,2). The internal transcribed spacer region (ITS1 and ITS2) of the ribosomal DNA and the β-tubulin gene were sequenced (GenBank Accession Nos. HQ909028 and JF826867, respectively) and compared with existing sequences using BLASTn. The ITS sequence shared 99% maximum identity with that of Ca. colhounii CBS 293.79 (GQ280565) from Java, Indonesia, and the β-tubulin sequence shared 97% maximum identity with that of Ca. colhounii CBS 114036 (DQ190560) isolated from leaf spots on Rhododendron sp. in North Carolina. The isolate was submitted to the Centraalbureau voor Schimmelcultures in the Netherlands (CBS 129628). To confirm pathogenicity, 5 ml of a conidial suspension (1 × 105/ml) were applied as a foliar spray or soil drench to four healthy ‘Bluecrop’ plants each in 10-cm plastic pots. Two water-sprayed and two water-drenched plants served as controls. Plants were misted intermittently for 2 days after inoculation. After 7 days at 25 ± 3°C, drench-inoculated plants developed necrotic, sporulating stem lesions at the soil line, while spray-inoculated plants showed reddish brown leaf and stem lesions. At 28 days, three drench-inoculated and one spray-inoculated plant had died, while others showed stem necrosis and wilting. No symptoms were observed on control plants. Fungal colonies reisolated from surface-disinfested symptomatic stem, leaf, and root segments appeared identical to the original isolate. Cy. colhounii was reported to cause a leaf spot on blueberry plants in nurseries in China (3), while Ca. crotalariae (Loos) D.K. Bell & Sobers (= Ca. ilicicola Boedijn & Reitsma) causes stem and root rot of blueberries in North Carolina (4). To our knowledge, this is the first report of Ca. colhounii causing a disease of blueberry in Michigan or the United States. Because of its destructive potential, this pathogen may pose a significant threat in blueberry nurseries. References: (1) P. W. Crous. Taxonomy and Pathology of Cylindrocladium (Calonectria) and Allied Genera. The American Phytopathological Society, St. Paul, MN, 2002. (2) L. Lombard et al. Stud. Mycol. 66:31, 2010. (3) Y. S. Luan et al. Plant Dis. 90:1553, 2006. (4) R. D. Milholland. Phytopathology 64:831, 1974.

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First report of Fusarium commune causing root and crown rot on maize in Italy

Plant Disease ◽

10.1094/pdis-01-21-0075-pdn ◽

2021 ◽

Author(s):

Monica Mezzalama ◽

Vladimiro Guarnaccia ◽

Ilaria Martino ◽

Giulia Tabome ◽

Maria Lodovica GULLINO

Keyword(s):

Plant Disease ◽

Tap Water ◽

Morphological Characteristics ◽

Plant Diseases ◽

Crown Rot ◽

Conidial Suspension ◽

Fusarium Spp ◽

First Report ◽

Root And Crown Rot ◽

Pathogenicity Tests

Maize (Zea mays L.) is a cereal crop of great economic importance in Italy; production is currently of 62,587,469 t, with an area that covers 628,801 ha, concentrated in northern Italy (ISTAT 2020). Fusarium species are associated with root and crown rot causing failures in crop establishment under high soil moisture. In 2019 maize seedlings collected in a farm located in San Zenone degli Ezzelini (VI, Italy) showed root and crown rot symptoms with browning of the stem tissues, wilting of the seedling, and collapsing due to the rotting tissues at the base of the stem. The incidence of diseased plants was approximately 15%. Seedlings were cleaned thoroughly from soil residues under tap water. Portions (about 3-5 mm) of tissue from roots and crowns of the diseased plants were cut and surface disinfected with a water solution of NaClO at 0.5% for 2 minutes and rinsed in sterile H20. The tissue fragments were plated on Potato Dextrose Agar (PDA) amended with 50 mg/l of streptomycin sulfate and incubated for 48-72 hours at 25oC. Over the 80 tissue fragments plated, 5% were identified as Fusarium verticillioides, 60% as Fusarium spp., 35% developed saprophytes. Fusarium spp. isolates that showed morphological characteristics not belonging to known pathogenic species on maize were selected and used for further investigation while species belonging to F. oxysporum were discarded. Single conidia of the Fusarium spp. colonies were cultured on PDA and Carnation Leaf Agar (CLA) for pathogenicity tests, morphological and molecular identification. The colonies showed white to pink, abundant, densely floccose to fluffy aerial mycelium. Colony reverse showed light violet pigmentation, in rings on PDA. On CLA the isolates produced slightly curved macronidia with 3 septa 28.1 - 65.5 µm long and 2.8-6.3 µm wide (n=50). Microconidia were cylindrical, aseptate, 4.5 -14.0 µm long and 1.5-3.9 µm wide (n=50). Spherical clamydospores were 8.8 ± 2.5 µm size (n=30), produced singly or in pairs on the mycelium, according to the description by Skovgaard et al. (2003) for F. commune. The identity of two single-conidia strains was confirmed by sequence comparison of the translation elongation factor-1α (tef-1α), and RNA polymerase II subunit (rpb2) gene fragments (O’Donnell et al. 2010). BLASTn searches of GenBank, and Fusarium-ID database, using the partial tef-1α (MW419921, MW419922) and rpb2 (MW419923, MW419924) sequences of representative isolate DB19lug07 and DB19lug20, revealed 99% identity for tef-1α and 100% identity to F. commune NRRL 28387(AF246832, AF250560). Pathogenicity tests were carried out by suspending conidia from a 10-days old culture on PDA in sterile H2O to 5×104 CFU/ml. Fifty seeds were immersed in 50 ml of the conidial suspension of each isolate for 24 hours and in sterile water (Koch et al. 2020). The seeds were drained, dried at room temperature, and sown in trays filled with a steamed mix of white peat and perlite, 80:20 v/v, and maintained at 25°C and RH of 80-85% for 14 days with 12 hours photoperiod. Seedlings were extracted from the substrate, washed under tap water, and observed for the presence of root and crown rots like the symptoms observed on the seedlings collected in the field. Control seedlings were healthy and F. commune was reisolated from the symptomatic ones and identified by resequencing of tef-1α gene. F. commune has been already reported on maize (Xi et al. 2019) and other plant species, like soybean (Ellis et al. 2013), sugarcane (Wang et al. 2018), potato (Osawa et al. 2020), indicating that some attention must be paid in crop rotation and residue management strategies. To our knowledge this is the first report of F. commune as a pathogen of maize in Italy. References Ellis M L et al. 2013. Plant Disease, 97, doi: 10.1094/PDIS-07-12-0644-PDN. ISTAT. 2020. http://dati.istat.it/Index.aspx?QueryId=33702. Accessed December 28, 2020. Koch, E. et al. 2020. Journal of Plant Diseases and Protection. 127, 883–893 doi: 10.1007/s41348-020-00350-w O’Donnell K et al. 2010. J. Clin. Microbiol. 48:3708. https://doi.org/10.1128/JCM.00989-10 Osawa H et al. 2020. Journal of General Plant Pathology, doi.org/10.1007/s10327-020-00969-5. Skovgaard K 2003. Mycologia, 95:4, 630-636, DOI: 10.1080/15572536.2004.11833067. Wang J et al. 2018. Plant Disease, 102, doi/10.1094/PDIS-07-17-1011-PDN Xi K et al. 2019. Plant Disease, 103, doi/10.1094/PDIS-09-18-1674-PDN

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First Report of Crown and Root Rot Caused by Pythium myriotylum on Hemp (Cannabis sativa) in Arizona

Plant Disease ◽

10.1094/pdis-12-20-2712-pdn ◽

2021 ◽

Author(s):

Jiahuai Hu

Keyword(s):

Root Rot ◽

Cannabis Sativa ◽

Morphological Characteristics ◽

Crown Rot ◽

Pathogenicity Test ◽

Light Cycle ◽

Pythium Myriotylum ◽

First Report ◽

Leaf Chlorosis ◽

Query Coverage

During August and September 2020, symptoms of leaf chlorosis, stunting, and wilting were observed on indoor hemp plants (Cannabis sativa L. cv. ‘Wedding Cake’) in a commercial indoor facility located in Coolidge, Arizona. Plants were grown in soilless coconut coir growing medium (Worm Factory COIR250G10), watered with 1.5 to 2.1 liters every 24 h through drip irrigation, and supplemented with 18 h of lighting. About 35% of plants displayed symptoms as described above and many symptomatic plants collapsed. To identify the causal agent, crown and root tissues from four symptomatic plants were harvested and rinsed with tap water. Tissue fragments (approx. 2 to 4 mm in size) were excised from the margins of the stem and root lesions, surface sterilized in 0.6% sodium hypochlorite for 1 min, rinsed well in sterile distilled water, blotted dry, and plated on potato dextrose agar (PDA) and on oomycete-selective clarified V8 media containing pimaricin, ampicillin, rifampicin, and pentachloronitrobenzene (PARP). Plates were incubated at room temperature (21-24 oC). Five isolates resembling Pythium were transferred after 3 days and maintained on clarified V8 media. Morphological characteristics were observed on grass blade cultures (Waterhouse 1967). Grass blades were placed on CV8 inoculated with the isolate. After a 1-day incubation at 25°C, the colonized blades were transferred to 8 ml of soil water extract in a Petri dish. Ten sporangia and oogonia were selected randomly and their diameters were measured under the microscope. Sporangia were mostly filamentous, undifferentiated or inflated lobulate, ranging from 7 to 17 µm in diameter. Knob-like appressoria were observed on branching clusters. Bulbous-like antheridia were formed on branched stalk with 1-8 antheridia per oogonium. Globose oogonia were terminal or intercalary and ranged from 21 to 33 µm in diameter. Globose oospores were mostly aplerotic and ranged from 15 to 21 μm in diameter. Based on these morphological characteristics, isolates were tentatively identified as Pythium myriotylum (Watanabe, 2002). Genomic DNA was extracted from mycelial mats of two isolates using DNeasy Plant Pro Kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s instructions. The internal transcribed spacer (ITS) region of rDNA was amplified with primers ITS1/ITS4 and two identical nucleotide sequences were obtained and deposited under accession number MW380925. A BLASTn search revealed ≥ 98% query coverage and 100% match with sequences HQ237488.1, KY019264.1, and KM434129, which were isolates of P. myriotylum from palm, tobacco, and ginger, respectively. To fulfill Koch’s postulates, pathogenicity tests were conducted with 2 isolates using plants of ‘Wedding Cake’ grown in 12 1.9-liter pots filled with a steam-disinfested potting mix (Sungro Professional Growing Mix). Pots were placed in a plastic container and watered to flooding three times a week. Plants were maintained in a greenhouse with 18 h/10 h day/night supplemental light cycle (15-28 oC). Plants were fertilized weekly with Peters Professional fertilizer at 1mg/ml. Four plants were inoculated with each isolate at three weeks after seed sowing by placing two 5-mm mycelial plugs from active growing 4 days-old cultures on PDA media adjacent to the main root mass at an approximately 3 cm depth. Four plants were inoculated with blank PDA plugs as controls. Symptoms of leaf chlorosis, crown rot and wilting were observed after four weeks while control plants remained symptomless. P. myriotylum was re-isolated from necrotic roots of inoculated plants after surface-sterilization, but not from control plants. The pathogenicity test was repeated once. While P. myriotylum often occurs in warmer regions and has a wide host range of >100 host plant species including numerous economically important crops (Wang et al., 2003), there are only two reports of this pathogen on indoor hemp plants in a greenhouse in Connecticut (McGehee et al., 2019) and in Canada (Punja et al., 2019). This is the first report of P. myriotylum causing root and crown rot of indoor hemp in Arizona. A more careful water management in soilless growth medium to reduce periods of saturation would minimize the risk of Pythium root rot in indoor hemp production.

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First report of Fusarium falciforme (FSSC 3+4) causing root rot on chickpea in Mexico

Plant Disease ◽

10.1094/pdis-05-21-1123-pdn ◽

2021 ◽

Author(s):

Sixto Velarde Felix ◽

Victor Valenzuela ◽

Pedro Ortega ◽

Gustavo Fierros ◽

Pedro Rojas ◽

...

Keyword(s):

Fusarium Solani ◽

Root Rot ◽

Species Complex ◽

Elongation Factor ◽

Morphological Characteristics ◽

Aerial Mycelium ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Single Spore ◽

First Report

Chickpea (Cicer aretinium L.) is a legume crop of great importance worldwide. In January 2019, wilting symptoms on chickpea (stunted grow, withered leaves, root rot and wilted plants) were observed in three fields of Culiacan Sinaloa Mexico, with an incidence of 3 to 5%. To identify the cause, eighty symptomatic chickpea plants were sampled. Tissue from roots was plated on potato dextrose agar (PDA) medium. Typical Fusarium spp. colonies were obtained from all root samples. Ten pure cultures were obtained by single-spore culturing (Ff01 to Ff10). On PDA the colonies were abundant with white aerial mycelium, hyphae were branched and septae and light purple pigmentation was observed in the center of old cultures (Leslie and Summerell 2006). From 10-day-old cultures grown on carnation leaf agar medium, macroconidias were falciform, hyaline, with slightly curved apexes, three to five septate, with well-developed foot cells and blunt apical cells, and measured 26.6 to 45.8 × 2.2 to 7.0 μm (n = 40). The microconidia (n = 40) were hyaline, one to two celled, produced in false heads that measured 7.4 to 20.1 (average 13.7) μm × 2.4 to 8.9 (average 5.3) μm (n = 40) at the tips of long monophialides, and were oval or reniform, with apexes rounded, 8.3 to 12.1 × 1.6 to 4.7 μm; chlamydospores were not evident. These characteristics fit those of the Fusarium solani (Mart.) Sacc. species complex, FSSC (Summerell et al. 2003). The internal transcribed spacer and the translation elongation factor 1 alpha (EF1-α) genes (O’Donnell et al. 1998) were amplified by polymerase chain reaction and sequenced from the isolate Ff02 and Ff08 (GenBank accession nos. KJ501093 and MN082369). Maximum likelihood analysis was carried out using the EF1-α sequences (KJ501093 and MN082369) from the Ff02 and Ff08 isolates and other species from the Fusarium solani species complex (FSSC). Phylogenetic analysis revealed the isolate most closely related with F. falciforme (100% bootstrap). For pathogenicity testing, a conidial suspension (1x106 conidia/ml) was prepared by harvesting spores from 10-days-old cultures on PDA. Twenty 2-week-old chickpea seedlings from two cultivars (P-2245 and WR-315) were inoculated by dipping roots into the conidial suspension for 20 min. The inoculated plants were transplanted into a 50-hole plastic tray containing sterilized soil and maintained in a growth chamber at 25°C, with a relative humidity of >80% and a 12-h/12-h light/dark cycle. After 8 days, the first root rot symptoms were observed on inoculating seedlings and the infected plants eventually died within 3 to 4 weeks after inoculation. No symptoms were observed plants inoculated with sterilized distilled water. The fungus was reisolated from symptomatic tissues of inoculated plants and was identified by sequencing the partial EF1-α gene again and was identified as F. falciforme (FSSC 3 + 4) (O’Donnell et al. 2008) based on its morphological characteristics, genetic analysis, and pathogenicity test, fulfilling Koch’s postulates. The molecular identification was confirmed via BLAST on the FusariumID and Fusarium MLST databases. Although FSSC has been previously reported causing root rot in chickpea in USA, Chile, Spain, Cuba, Iran, Poland, Israel, Pakistan and Brazil, to our knowledge this is the first report of root rot in chickpea caused by F. falciforme in Mexico. This is important for chickpea producers and chickpea breeding programs.

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First Report of Crown Rot of Banana Caused by Fusarium porliferatum in Georgia, USA

Plant Disease ◽

10.1094/pdis-08-21-1809-pdn ◽

2021 ◽

Author(s):

Sumyya Waliullah ◽

Greg E. Fonsah ◽

Jason Brock ◽

Yonggang Li ◽

Emran Ali

Keyword(s):

Sequence Similarity ◽

Morphological Characteristics ◽

Fusarium Proliferatum ◽

Crown Rot ◽

Post Inoculation ◽

Conidial Suspension ◽

Banana Fruit ◽

Single Spore ◽

First Report ◽

Initial Symptoms

Crown rot is one of the most damaging disease of banana fruit characterized by rot and necrosis of crown tissues. In severe cases, the disease can spread to the pedicel and banana pulp. Crown rot can be infected by several common fungi, including Lasiodiplodia theobromae, Musicillium theobromae, Colletotrichum musae, and a complex of Fusarium spp. and lead to softening and blackening of tissues (Lassois et al., 2010; Kamel et al., 2016; Triest et al., 2016; Snowdon, 1990). In November 2020, typical crown rot of banana fruits (cv. Pisang Awak, belonging to the tetraploid AABB genome) were observed from UGA Banana Research 12 Plots, Tifton, GA, with incidence rates of 15%. Initial symptoms appeared in the infected crown of green banana fruits. As the infection progressed, the crown tissues became blackened and softened, followed by an internal development of infection affecting the peduncle and the fruit, triggered early ripening of bananas. At last, the development of necrosis on the pedicels and fruits appeared and caused the fingers to fall off. To identify the pathogen, tissue pieces (~0.25 cm2) from the infected crown and pedicles were surface-sterilized in a 10% bleach solution for 1 min, followed by 30 s in 70% EtOH. The disinfected tissues were rinsed in sterile water 3 times and cultured on potato dextrose agar (PDA) amended with 50 µg/ml streptomycin at 25°C in the dark for 5–10 days. Isolates of the pathogen were purified using the single-spore isolation method (Leslie and Summerell 2006). Colonies on PDA produced fluffy aerial mycelium and developed an intense purple pigment when viewed from the underside. A range of colony pigmentation and growth rates were observed among the isolates. The microconidia were ovoid, hyaline, or ellipse in shape. The morphological features of the isolates were identified as Fusarium proliferatum (Leslie and Summerell, 2006). To further identify the isolates, genomic DNA was extracted from a representative isolate. And the internal transcribed spacer (ITS) region, the partial elongation factor (TEF1-α) gene and the β-tubulin gene (TUB2)were amplified and sequenced using the primers ITS1/ITS4 (Yin et al. 2012), EF-1 /EF-2 (O’Donnell et al. 1998) and B-tub1 /B-tub2 (O’Donnell and Cigelnik, 1997), respectively. The amplicons were sequenced and deposited in NCBI (accessions no. MZ292989, MZ293071 for ITS: MZ346602, MZ346603 for TEF1-α and MZ346600 and MZ346601 for B-tub). The ITS, TEF1-α, and B-tub sequences of the isolates showed 100% sequence similarity with Fusarium proliferatum isolates (accessions no. MT560212, LS42312, and LT575130, respectively) using BLASTn in Genbank. For pathogenicity testing, three whole bunched bananas sterilized with 10% bleach solutions and washed by sterilized water, were cut into 5 bananas per brunch. The cut surface of the banana crown was inoculated with conidial suspension (1.0 × 107 cfu/ml) of the pathogen with pipette tips. Equal number of bananas were treated with sterilized water in the same volume as a control. All bananas were sealed in a plastic bag and incubated at 25°C. After 7 days post inoculation, all inoculated bananas showed initial crown rot symptoms while no symptoms were observed on the control bananas. The fungus was re-isolated from the symptomatic tissues of infected bananas and confirmed to be genetically identical to F. proliferatum of the original inoculated strains according to morphological characteristics and molecular identification, fulfilling Koch’s postulates. To the best of our knowledge, this is the first report of F. proliferatum causing crown rot on bananas in Georgia, USA.

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First report of root rot of Schisandra chinensis caused by Fusarium acuminatum in Northeast China

Plant Disease ◽

10.1094/pdis-05-21-1052-pdn ◽

2021 ◽

Author(s):

Baoyu Shen ◽

Wensong Sun ◽

Kun Liu ◽

Jing Tian Zhang

Keyword(s):

Root Rot ◽

Apical Cell ◽

Morphological Characteristics ◽

Liaoning Province ◽

Effective Control ◽

Distilled Water ◽

Conidial Suspension ◽

Schisandra Chinensis ◽

First Report ◽

Fusarium Acuminatum

Wuweizi [Schisandra chinensis（Turcz.）Baill.] is used for traditional medicine in northeastern China. In August of 2019, root rot of S. chinensis with an incidence of 30%-50% was observed in a commercial field located in Liaozhong city (41º29’57” N, 122º52’33” E) in the Liaoning province of China. The diseased plants were less vigorous, stunted, and had leaves that turned yellow to brown. Eventually, the whole plant wilted and died. The diseased roots were poorly developed with brown lesion and eventually they would rot. To determine the causal agent, symptomatic roots were collected, small pieces of root with typical lesions were surface sterilized in 2% NaOCl for 3 min, rinsed three times in distilled water, and then plated onto PDA medium. After incubation at 26°C for 5 days, whitish-pink or carmine to rose red colonies on PDA were transferred to carnation leaf agar (CLA). Single spores were isolated with an inoculation needle using a stereomicroscope. Five single conidia isolates obtained from the colonies were incubated at 26°C for 7 days, abundant macroconidia were formed in sporodochia. Macroconidia were falcate, slender, with a distinct curve to the latter half of the apical cell, mostly 3 to 5 septate, measuring 31.3 to 47.8 × 4.8 to 7.5µm (n=50). Microconidia were oval and irregular ovals, 0-1 septate, measuring 5.0 to 17.5 × 2.5 to 17.5µm (n=50). Chlamydospores formed in chains on within or on top of the mycelium. Morphological characteristics of the isolates were in agreement with Fusarium acuminatum (Leslie and Summerell, 2006). To confirm the identity, the partial sequence of the translation elongation factor 1 alpha (TEF1-á) gene of five isolates was amplified using the primers EF-1(ATGGGTAAGGARGACAAG) and EF-2 (GGARGTACCAGTSATCATGTT) (O’Donnell et al. 2015 ) and sequenced. The rDNA internal transcribed spacer (ITS) region for the five isolates was also amplified using the primers ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTATTGATATGC) (White et al.1990) and sequenced. The identical sequences were obtained, and one representative sequence of isolate WW31-5 was submitted to GenBank. BLASTn analysis of the TEF-á sequence (MW423624) and ITS sequence (MZ145386), revealed 100%（708/685bp, 563/563bp）sequence identity to F. acuminatum MH595498 and MW560481, respectively. Pathogenicity tests were conducted in greenhouse. Inoculums of F. acuminatum was prepared from the culture of WW31-5 incubated in 2% mung beans juice on a shaker (140 rpm) at 26°C for 5 days. Ten roots of 2-years old plants of S. chinensis were immersed in the conidial suspension (2 × 105 conidia/ml) for 6 hours, and another ten roots immersed in sterilized distilled water in plastic bucket for 6 hours. All these plants were planted into pots with sterilized field soil (two plants per pot). Five pots planted with inoculated plants and another five pots planted with uninoculated plants served as controls. All ten pots were maintained in a greenhouse at 22-26°C for 21 days and irrigated with sterilized water. The leaves of the inoculated plants became yellow，gradually dried up, eventually finally all the aboveground parts died. The roots of the inoculated plants were rotted. Non-inoculated control plants had no symptoms. F. acuminatum was reisolated from the roots of inoculated plants and had morphology identical to the original isolate. The experiment was repeated twice with similar results. F. acuminatum has been reported as a pathogen caused root rot of ginseng (Wang et al. 2016) and not reported on Wuweizi in China. To our knowledge, this is the first report of root rot of S. chinensis caused by F. acuminatum. We have also observed the disease at Benxi city of Liaoning Province in 2020 and it has become an important disease in production of S. chinensis and the effective control method should be adopted to reduce losses.

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First Report of Berkeleyomyces basicola Causing Black Root Rot on Lisianthus (Eustoma grandiflorum) in China

Plant Disease ◽

10.1094/pdis-08-21-1724-pdn ◽

2021 ◽

Author(s):

Yishuo Huang ◽

Xuewen Xie ◽

Yanxia Shi ◽

A LI CHAI ◽

Lei Li ◽

...

Keyword(s):

Root Rot ◽

Mangifera Indica ◽

Large Subunit ◽

Morphological Characteristics ◽

Internal Transcribed Spacers ◽

Likelihood Method ◽

Conidial Suspension ◽

Eustoma Grandiflorum ◽

Black Root Rot ◽

First Report

Lisianthus (Eustoma grandiflorum (Raf.) Shinn.) is an important ornamental plant ranking in the top 10 cut flowers worldwide (Xiao et al., 2018). In 2020 and 2021, black root rot was found as a major disease limiting lisianthus production in Yunnan Province, China. Black root rot was first observed in early July 2020 on lisianthus grown in a commercial flower-production plantation, with nearly 60% plants infected. Symptoms appeared as coalescing necrotic lesions leading to black discoloration of the roots. Root damage induced by disease resulted in insufficient water and nutrient uptake by the plant, causing stunting and whole-plant wilting. The pathogen could not infect the intact endodermis, and vascular tissues below the discolored cortical tissue remained healthy. Symptomatic roots were surface sterilized using 1% NaClO for 1 min, rinsed three times in sterile water, placed onto potato dextrose agar (PDA), and incubated at 25°C for 7 days in the dark. The morphological characteristics were basically consistent: the colonies were white to gray in color, and the conidiophores were colorless to brown, solitary or clustered. Conidia were single-celled, colorless, rod-shaped, and obtuse at both ends. Chlamydospores were dark brown, clustered or solitary. The morphological characteristics of the pathogen were similar to those of Berkeleyomyces basicola (Berk. & Broome) W.J. Nel, Z.W. de Beer, T.A. Duong & M.J. Wingf. (Nakane et al. 2019). DNA was extracted from mycelia of representative isolate TB using the Plant Genomic DNA Kit (Tiangen, Beijing, China). The internal transcribed spacers (ITS), DNA replication licensing factor (MCM7), ribosomal large subunit (LSU), and 60S ribosomal protein RPL10 (60S) regions were amplified with primer pairs ITS1/ITS4 (Groenewald et al. 2013), MCM7-for/MCM7-rev, LR0R/LR5, and 60S-506F/60S-908R, respectively (Nel et al. 2018). Phylogenetic analysis of multiple genes (Bakhshi et al. 2018) was conducted with the maximum likelihood method using MEGA7. The sequences of our isolate (TB) and three published sequences of B. basicola were clustered into one clade with a 100% bootstrapping value. The accession numbers of B. basicola reference sequences are MF952423 (ITS), MF967079 (MCM7), MF948658 (LSU), and MF967072 (60S) of isolate CMW6714; MF952428 (ITS), MF967088 (MCM7), MF948661 (LSU), and MF967073 (60S) of isolate CMW25440; MF952429 (ITS), MF967102 (MCM7), MF948659 (LSU), and MF967075 (60S) of isolate CMW49352. The sequences of TB have been deposited in GenBank with accession numbers MZ351733 for ITS, MZ695817 for MCM7, MZ695816 for LSU, and MZ695815 for the 60S region. To verify the pathogenicity of the fungus, inoculations were performed on ten 2-month-old potted lisianthus plants by dipping the roots into a conidial suspension (105 spores/ml) for 2 h. Ten plants were mock inoculated with distilled water as a control. Symptoms of black root rot were observed 30 days after inoculation, whereas the control roots remained healthy. The causal fungus has a host range of over 230 species and is a destructive pathogen of many crops and ornamental plants, including cotton (Gossypium barbadense L.), tobacco (Nicotiana tabacum L.) and mango (Mangifera indica L.) (Shukla et al. 2021; Toksoz and Rothrock 2009). This is the first report worldwide of B. basicola infecting lisianthus. This discovery is of great importance for Chinese flower growers because this fungus is well established in the observed area, and effective measures are needed to manage this disease.

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First Report of Paraphoma chrysanthemicola Causing Crown and Leaf Rot of Atractylodes lancea in China

Plant Disease ◽

10.1094/pdis-11-21-2408-pdn ◽

2022 ◽

Author(s):

Hongyang Wang ◽

Chuanzhi Kang ◽

Wang Yue-Feng ◽

Sheng Wang ◽

Zhang Yan ◽

...

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

Crown Rot ◽

Post Inoculation ◽

Conidial Suspension ◽

Sterile Water ◽

First Report ◽

Atractylodes Lancea ◽

Detached Leaves ◽

Leaf Rot

Atractylodes lancea is an important traditional Chinese medicinal plant whose rhizome is used for treating complaints such as rheumatic diseases, digestive disorders, night blindness and influenza. Jiangsu Province is the optimal cultivation location for high-quality A. lancea rhizome. Since June 2019, symptoms of crown rot and leaf rot were observed in about 10-20% of the A. lancea in a plantation (31° 36' 1" N, 119° 6' 40" W) in Lishui, Jiangsu, China. Lesions occurred on the stem near the soil line and on the leaves (Fig. 1A). Disease incidence reached approximately 80-90% by September, 2021 (Fig. 1B) and resulted in severe loss of rhizome and seed yields. For pathogen isolation, ten samples of symptomatic stem segments and ten diseased leaves were collected, surface-sterilized using 5% NaClO solution, rinsed with sterile water, cut into 0.5-2 cm segments, and plated to potato dextrose agar (PDA), and then incubated at 30°C in darkness. Pure cultures of four isolates showing morphological characteristics of Paraphoma spp. were obtained, identified as a single P. chrysanthemicola strain, and named LSL3f2. Newly formed colonies initially consisted of white mycelia; the five-day-old colonies developed a layer of whitish grey mycelia with a grey underside. 20-day-old colonies had white mycelium along the margin and with a faint yellow inner circular part with irregular radial furrows, and the reverse side looking caramel and russet (Fig. 1C). Pycnidia were subglobose (diameter: 5 to 15 μm; Fig. 1D). Unicellular, bicellular or strings of globose or subglobose chlamydospores developed from hyphal cells (Fig. 1E and 1F). The internal transcribed spacer (ITS) region and large subulin-28S of LSL3f2 were cloned using primers ITS1/ITS4 and LR0R/LR7 (Aveskamp et al. 2010, Li et al. 2013), and deposited in GenBank (OK559658 and OK598973, respectively). BLASTn search and phylogenetic analysis showed the highest identity between LSL3f2 and P. chrysanthemicola sequences (Fig. 1G) and confirmed LSL3f2 as P. chrysanthemicola. Koch’s postulates were completed using one-month-old vegetatively propagated A. lancea plantlets growing on autoclaved vermiculite/peat mixture at 26°C with a light/dark cycle of 12/12 hours. Each plantlet was inoculated with 5 ml of conidial suspension in water (1 × 108 cfu/ml) by applying to soil close to the plantlet, with sterile water used as a mock control (n = 10). By 20 days post-inoculation, inoculated plantlets showed a range of disease symptoms consistent to those observed in infested fields (Fig. 1H). Pathogenicity was additionally confirmed using detached leaves inoculated with a colonized agar plug of LSL3f2 or an uninoculated control comparison (diameter = 5 mm) and incubated at 26℃ in the dark. Five to seven days post-inoculation, detached leaves showed leaf rot symptoms including lesions, yellowing and withering consistent with those in infested fields, while control leaves remained healthy (n = 10, Fig. 1I). The pathogen was reisolated from the diseased plantlets and detached leaves, in both cases demonstrating the micromorphological characteristics of LSL3f2. P. chrysanthemicola has been reported to cause leaf and crown rot on other plants such as Tanacetum cinerariifolium (Moslemi et al. 2018), and leaf spot on A. japonicain (Ge et al. 2016). However, this is the first report of P. chrysanthemicola causing crown and leaf rot on A. lancea in China.

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First report of Plectosphaerella cucumerina causing root rot on fennel in China

Plant Disease ◽

10.1094/pdis-01-21-0166-pdn ◽

2021 ◽

Author(s):

Fengfeng Cai ◽

Chengde Yang ◽

Ting Ma

Keyword(s):

Root Rot ◽

Morphological Characteristics ◽

Control Measures ◽

Effective Control ◽

Herbaceous Plant ◽

Conidial Suspension ◽

Foeniculum Vulgare ◽

Medicinal Uses ◽

First Report ◽

Plectosphaerella Cucumerina

Fennel (Foeniculum vulgare Mill.) is herbaceous plant commonly cultivated for culinary and medicinal uses in China (Shi et al. 2016 ). In May 2019, disease of fennel was observed in Yumen City, Gansu Province, China (N 40°28'/E 97°05'). The incidence across the fields (about 0.23 hectare) was about 4.5%. The outer leaves of diseased fennel wilted, the rhizome changed color from brown to dark brown，necrosis and rot symptoms developed on the root. Finally, the whole plant wilted and died. When pulling up, it was easy to break the root. To identify the pathogen, 15 samples of diseased plants were collected and symptomatic rhizome tissues were surface disinfected with 0.1% HgCl solution for 30 s, rinsed in sterilized water 3 times, placed on potato dextrose agar (PDA), and incubated at 25℃ in the dark. About 7 days, hyphal tips from the tissue edge were transferred to a new PDA for purification. Three isolates obtained were named as hxa, hxb and hxc. To confirm their pathogenicity, two-month old fennel seedings planted in pots, three seedings per pot, with sterilized nutrient soil were inoculated by pouring 50 ml of conidial suspension (107 conidium/mL) produced from the three isolates. Plants inoculated with sterilized water only were included as controls. Six pots of inoculated plants were maintained in climatic cabinet / chamber (> 85% RH, 25°C). The pathogenicity tests were conducted twice. After 7 days, the plants inoculated with conidial suspension of hxa developed brown necrosis and wilt symptoms resembling those originally observed in the field, whereas the controls and the plants inoculated with the other two isolates had no symptoms. Furthermore, hxa was reisolated from rhizome of these diseased plants. The results indicated that isolate hxa was the pathogen causing root rot of fennel. The colonies of hxa on PDA were white-to-cream, slimy, mycelium appressed, aerial mycelium absent. Mycelium was hyaline, septate and formed hyphal coils. Conidiophores were solitary, hyaline, sometimes crooked or sinuous, widest at the base, gradually tapering to the apex. Conidia were smooth, hyaline, aseptate, elliptical and ovoid, measuring 5.97 to 9.51 × 2.13 to 3.58 um (avg. 7.58×2.78, n=100). These morphological characters of the fungal isolates were identical to those of Plectosphaerella cucumerina (Carlucci et al. 2012). For molecular identification, genomic DNA was extracted from the mycelium, and the rDNA internal transcribed spacer (ITS) region, portions of the 28S ribosomal RNA (LSU), calmodulin (CaM) and translation elongation factor 1α (Ef‐1α) gene were amplified using primer pairs ITS1/ITS4, LROR/LR5, CMD5/CMD6 and 688f/1251R (White et al., 1990; Hopple et al., 1999; Hong et al., 2005; Alves et al., 2008), respectively. The sequences of these genes were deposited in GenBank (accessions: ITS as MW426266, LSU as MW433724, CaM as MW448071 and EF-1a as MW459981) and used in analysis to generate a phylogenetic tree. These sequences showed 100, 100, 96 and 97.32% homology to the sequences of P. cucumerina (GenBank accession no. EU594566, MH867359, KY416911 and KY964491), respectively. According to morphological characteristics and phylogenetic analysis, isolate hxa was identified as P. cucumerina. To our knowledge, this is the first report of P. cucumerina causing root rot of fennel in China as well as worldwide. This finding may help to take effective control measures of root rot on fennel.

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First Report of Crown Rot of Grafted Cucumber Caused by Fusarium solani in China

Plant Disease ◽

10.1094/pdis-03-10-0217 ◽

2010 ◽

Vol 94 (11) ◽

pp. 1377-1377 ◽

Cited By ~ 2

Author(s):

B.-J. Li ◽

Y. Liu ◽

Y.-X. Shi ◽

X.-W. Xie ◽

Y.-L. Guo

Keyword(s):

Disease Incidence ◽

Apical Cell ◽

Morphological Characteristics ◽

Crown Rot ◽

Liaoning Province ◽

Economic Losses ◽

Peat Moss ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

First Report

Grafting has been widely and effectively used in cucumber (Cucumis sativus) cultivation for approximately 30 years in China to avoid Fusarium wilt caused by Fusarium oxysporum Schl. f. sp. cucumerinum Owen. In greenhouses, 90% of cucumbers are grafted onto pumpkin (Cucurbita moschata) rootstock. However, in March 2009, a severe crown rot causing yellowing and wilting of the leaves was observed on grafted cucumber in a large number of greenhouses in Lingyuan, western Liaoning Province in China. Symptoms consisted of dark brown, water-soaked lesions and a dense, white mycelial mat at the base of the stem. Lingyuan is the largest district for cucumber cultivation using grafting techniques in solar greenhouses in China. In 30 surveyed greenhouses in Sanshijiazi Village in the city of Lingyuan, the incidence of affected plants ranged from 10 to 40%, which caused serious economic losses. Fusarium spp. were isolated from the surface-sterilized basal stems of symptomatic plants on potato dextrose agar and incubated on potato sucrose agar for 4 days at 25°C. Colonies of the isolates produced a brown pigmentation and sparse, aerial mycelia, with a cream color on the underside. Conidiophores were elongated and branched or unbranched. Microconidia were abundant, hyaline, ellipsoid to ovoid, and 6 to 14 × 2.5 to 3.5 μm. Macroconidia were cylindrical, abundant, mostly two to six septate, 22 to 63 × 3.2 to 5.0 μm, with the apical cell rounded and blunt, and the basal cell rounded. On the basis of morphological characteristics, the fungus was identified as F. solani (C. Booth). For confirmation, the internal transcribed spacer region of rDNA was amplified and sequenced. A 449-bp sequence shared 99% homology with that of a F. solani GenBank accession previously reported from Japan (No. AF150473.1). The new sequence was deposited in GenBank (Accession No. HM015882). Pathogenicity of three isolates was determined in two experiments using different methods of inoculation. In one, 30 seedlings of pumpkin (C. moschata) with one true leaf each were inoculated by dipping their roots in a suspension of 106 spores ml–1, while control plants were mock inoculated with sterile water. Plants were then potted in a sterile mix of peat moss and vermiculite (2:1 vol/vol). In the other, pregerminated pumpkin seeds were sown in the same medium with a conidial suspension added at a rate of 106 spores ml–1, while other seeds were sown in sterile soil as controls. Plants for both experiments were maintained in a greenhouse at 25°C. Twelve days after inoculation, inoculated plants in both experiments showed a cortical rot on the crown and stem base with a brown, water-soaked appearance. Twenty-one days later, inoculated plants developed wilting and yellowed leaves. Disease incidence was 100%. No symptoms occurred on the control plants. Both experiments were repeated once with the same results. The pathogen was recovered from symptomatic tissue, confirming Koch's postulates. F. solani has been previously reported to cause root rot on cucurbit in California (2) and crown rot on grafted cucumber in the Netherlands (1). To our knowledge, this is the first report of crown rot of grafted cucumber caused by F. solani in China. References: (1) L. C. P. Kerling and L. Bravenboer. Neth. J. Plant Pathol. 73:15, 1967. (2) T. A. Tousson and W. C. Snyder. Phytopathology 51:17, 1961.

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