First report of Diaporthe eres causing root rot of Coptis chinensis Franchet

Chongqing coptis (Coptis chinensis Franchet) industry produces more than 60% of the Chinese coptis crop, and has been exported to many countries and regions. Since 2008, root rot has become a serious and widespread disease on coptis plants in Shizhu county with an average incidence of 40%, and yield losses up to 67%. Symptomatic coptis plants showed stunted growth, with the fibrous roots and main roots having brown or black, rotten, necrotic lesions. To our knowledge, Fusarium solani, F. carminascens, F. oxysporum and F. tricinctum have been previously reported as pathogens of coptis root rot (Luo et al. 2014; Cheng et al. 2020; Wu et al. 2020), but non Fusarium pathogens has not been reported yet. In order to identify new pathogens, 33 diseased roots were collected from Shizhu (30°18'N, 108°30'E) in October 2019. Small samples (0.5 cm in length) were cut from the border between diseased and healthy tissue, and then put on PDA after surface sterilization. Cultures were incubated at 25°C in dark until fungal colonies were observed. After subculturing for 3 times, 3 out of 21 isolates yielded a similar type of fungal colony. White, aerial, fluffy mycelium were formed and reached 8.3 cm diameter within 7 days, and dark pigmentation developed in the centre. Colonies turned to gray with age, and abundant dark brown pycnidia and black stromata were formed at maturity. Alpha conidia were aseptate, hyaline, fusiform to ellipsoidal, often biguttulate, measuring (6.0-8.5)×(2.0-3.0) μm. Beta conidia were aseptate, hyaline, linear to hooked, measuring (18-30)×(1.0-1.5) μm (Figure S1). For further identification, a multigene phylogenetic analysis was carried out. The internal transcribed spacer (ITS), translation elongation factor 1ɑ (tef1-ɑ), histone H3 (his3), calmodulin (cal), and β-tubulin (tub2) gene regions were amplified with ITS1/ITS4, EF1-728F/EF1-986R, CYLH3F/H3-1b, CAL228F/CAL737R, T1/Bt2b (White et al. 1990; Glass and Donaldson 1995; Carbone and Kohn 1999; Crous et al. 2004). GenBank accession numbers of isolate H13 were MT463391 for the ITS region, MT975573 for tef1-ɑ, MT975574 for his3, MT975575 for cal, and MT975576 for tub2. BLAST results showed the ITS, tef1-ɑ, his3, cal and tub2 sequences revealed 99.82% (553/554 base pairs), 100% (347/347 base pairs), 100% (474/474 base pairs), 99.39% (486/489 base pairs), and 99.14% (803/810 base pairs) homology respectively with those of Diaporthe eres (MN816416.1, KU557616.1, KC343564.1, KU557595.1, and KY569366.1). Thus, H13 were identified as D. eres based on its morphological and molecular characteristics. Pathogenicity of D. eres in coptis was investigated using the H13 isolate (1 of the 3 isolates). The roots of 10 healthy 2-year-old coptis plants were individually inoculated with 5 ml of a 106 conidia/mL conidial suspension and sterilized water was used to mock inoculate. Thirty days after inoculation, most of the inoculated coptis roots showed dark brown and rotten root, similar to those observed in the field, whereas mock inoculated roots showed healthy. D. eres was recovered from symptomatic roots and identified based on morphology. To our knowledge, this is the first report of D. eres causing root rot of coptis not only in China but anywhere in the world.

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First Report of Fusarium solani Causing Root Rot on Coptis chinensis in Southwestern China

Plant Disease ◽

10.1094/pdis-02-14-0164-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1273-1273 ◽

Cited By ~ 2

Author(s):

X.-M. Luo ◽

J.-L. Li ◽

J.-Y. Dong ◽

A.-P. Sui ◽

M.-L. Sheng ◽

...

Keyword(s):

Fusarium Solani ◽

Root Rot ◽

Southwestern China ◽

Molecular Characteristics ◽

Distilled Water ◽

Conidial Suspension ◽

Storage Roots ◽

Coptis Chinensis ◽

Causal Organism ◽

First Report

China is the world's largest producer country of coptis (Coptis chinensis), the rhizomes of which are used in traditional Chinese medicine. Since 2008, however, root rot symptoms, including severe necrosis and wilting, have been observed on coptis plants in Chongqing, southwestern China. Of the plants examined from March 2011 to May 2013 in 27 fields, 15 to 30% were covered with black necrotic lesions. The leaves of infected plants showed wilt, necrotic lesions, drying, and death. The fibrous roots, storage roots, and rhizomes exhibited brown discoloration and progressive necrosis that caused mortality of the infected plants. Infected plants were analyzed to identify the causal organism. Discoloration of the internal vascular and cortical tissues of the rhizomes and taproots was also evident. Symptomatic taproots of the diseased coptis were surface sterilized in 1% sodium hypochlorite for 2 min, rinsed in sterile distilled water for 2 min, and then air-dried in sterilized atmosphere/laminar flow. Small pieces of disinfested tissue (0.3 cm in length) were transferred to petri dishes containing potato dextrose agar (PDA) supplemented with 125 μg ml–1 streptomycin sulfate and 100 μg ml–1 ampicillin, and incubated for 5 days at 25°C with a 12-h photoperiod. Four distinct species of fungal isolates (HL1 to 4) derived from single spores were isolated from 30 plants with root rot symptoms collected from the study sites. To verify the pathogenicity of individual isolates, healthy coptis plants were inoculated by dipping roots into a conidial suspension (106 conidia/ml) for 30 min (15 plants per isolate), as described previously (1). Inoculated plants were potted in a mixture of sterilized quartz sand-vermiculite-perlite (4:2:1, v/v) and incubated at 25/18°C and 85 to 90% relative humidity (day/night) in a growth chamber with a daily 16-h photoperiod of fluorescent light. Plants dipped in sterile distilled water were used as controls. After 15 days, symptoms similar to those observed in the field were observed on all plants (n = 15) that were inoculated with HL1, but symptoms were not observed on plants inoculated with HL2, HL3, and HL4, nor on control plants. HL1 was re-isolated from symptomatic plants but not from any other plants. Morphological characterization of HL1 was performed by microscopic examination. The septate hyphae, blunt microconidia (2 to 3 septa) in the foot cell and slightly curved microconidia in the apical cell, and chlamydospores were consistent with descriptions of Fusarium solani (2). The pathogen was confirmed to be F. solani by amplification and sequencing of the ribosomal DNA internal transcribed spacer (rDNA-ITS) using the universal primer pair ITS4 and ITS5. Sequencing of the PCR product revealed a 99 to 100% similarity with the ITS sequences of F. solani in GenBank (JQ724444.1 and EU273504.1). Phylogenetic analysis (MEGA 5.1) using the neighbor-joining algorithm placed the HL1 isolate in a well-supported cluster (97% bootstrap value based on 1,000 replicates) with JQ724444.1 and EU273504.1. The pathogen was thus identified as F. solani based on its morphological and molecular characteristics. To our knowledge, this is the first report of root rot of coptis caused by F. solani in the world. References: (1) K. Dobinson et al. Can. J. Plant Pathol. 18:55, 1996. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, 2006.

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First Report of Boeremia Blight Caused by Boeremia exigua var. exigua on Pyrethrum in Australia

Plant Disease ◽

10.1094/pdis-05-11-0424 ◽

2011 ◽

Vol 95 (11) ◽

pp. 1478-1478 ◽

Cited By ~ 5

Author(s):

S. J. Jones ◽

F. S. Hay ◽

T. C. Harrington ◽

S. J. Pethybridge

Keyword(s):

Disease Incidence ◽

Elongation Factor ◽

Its Region ◽

Tween 20 ◽

Width Ratio ◽

Malt Extract ◽

Base Pairs ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Surface Sterilization

Pyrethrum (Tanacetum cinerariifolium) is produced for extraction of insecticidal compounds from the flower achenes. In 2004 and 2006, isolations from necrotic lesions on stems and leaves in three fields in northern Tasmania, Australia yielded four unidentified fungal isolates. Leaf lesions were medium brown and circular (2 to 4 mm in diameter) or irregular in shape (2 to 5 mm long). Stem lesions were irregular, necrotic spots, 5 to 15 mm below the flower peduncle, medium brown, 2 to 4 mm long, and 1 to 2 mm wide. Isolations were conducted on water agar following surface sterilization. Isolates were identified by colony characteristics and the presence of metabolite ‘E’ (1). On oatmeal agar (OA), colonies had irregular margins, were greenish olivaceous-to-olivaceous gray with sparse, white, floccose, aerial mycelia. On malt extract agar (MEA), cultures were variable in color with olivaceous black centers with soft, dense, aerial mycelia. Conidia were hyaline, ellipsoidal to oblong, mainly aseptate, but occasionally 1-septate with dimensions ranging from 2.5 to 7.5 × 1.8 to 3.8 μm (length/width ratio = 1.7 to 2.1). All isolates had moderate reactions to the NaOH test for metabolite ‘E’. DNA was extracted from all four isolates with a DNeasy Plant Mini Kit (QIAGEN Inc., Valencia, CA). For identification, the internal transcribed spacer region (ITS1, 5.8s, and ITS2) and part of the translation elongation factor (TEF) region were amplified and sequenced. Primers ITS1 and ITS4 (2) were used for the ITS region and primers EFCF1 (5′-AGTGCGGTGGTATCGACAAG) and EFCF6 (3′-CATGTCACGGACGGCGAAAC) were used for the TEF. Amplicons were sequenced in both directions and consensus sequences assembled. The ITS sequence was 100% identical to Boeremia exigua var. exigua (GenBank Accession No. GU237715). Base pairs 413 to 1,214 of the TEF sequence from the pyrethrum isolates matched base pairs 1 to 802 (799 of 802 identities) of B. exigua var. exigua (GenBank Accession No. GU349080). All isolates were confirmed as B. exigua var. exigua using morphology and sequencing. Pathogenicity tests were conducted three times in separate glasshouse trials for two of the four isolates. For each isolate, conidial suspensions in water (3 ml/plant) from MEA, adjusted to 5 × 105/ml were applied with Tween 20 (1 drop per 100 ml of water) to 8-week-old pyrethrum plants (five pots per isolate with four plants per pot) using a hand-held spray bottle. Twenty plants were sprayed with water and Tween 20 as nontreated controls. Plants were covered with plastic bags for 48 h after inoculation and examined for symptoms after 15 days at 20°C. Disease incidence (number of symptomatic leaves affected per total number of leaves) of the inoculated plants varied from 7.5 to 9.4%. Noninoculated plants did not develop symptoms. Isolations resulted in cultures morphologically identical on MEA and OA to those inoculated. To our knowledge, this is the first report of B. exigua var. exigua causing disease in pyrethrum. Cultures were deposited in the New South Wales Department of Agriculture collection (DAR79101 to 79104) and TEF and ITS sequences for DAR79101 in GenBank (Accession Nos. JF925328 and JF925329, respectively). Boeremia blight is likely to contribute to the fungal disease complex causing reductions in green leaf area in Australian pyrethrum production. References: (1) M. M. Aveskamp et al. Stud. Mycol. 65:1, 2010. (2) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

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First Report of Lily Blight and Wilt Caused by Fusarium tricinctum in China

Plant Disease ◽

10.1094/pdis-11-12-1010-pdn ◽

2013 ◽

Vol 97 (7) ◽

pp. 993-993 ◽

Cited By ~ 10

Author(s):

Y. Y. Li ◽

Y. J. Wang ◽

Z. K. Xie ◽

R. Y. Wang ◽

Y. Qiu ◽

...

Keyword(s):

Root Rot ◽

Elongation Factor ◽

Aerial Mycelium ◽

Its Region ◽

Universal Primers ◽

Conidial Suspension ◽

Root Tips ◽

First Report ◽

Horticultural Crops ◽

Blastn Analysis

Lily (Lilium spp.) is one of the most well-known horticultural crops, and plays an important economic role in China. In September 2011, wilted plants were observed on Lilium oriental hybrid cultivar ‘Sorbonne’ growing in Longde County, Ningxia Hui Autonomous Region, China. Disease symptoms included wilting, stem and root rot, brown spots of bulbs and then bulbs rotting and spalling from the basal disc, plus a progressive yellowing and defoliation of the leaves from the base. Diseased plants were sampled from fields. Small pieces of symptomatic bulbs, stems, and roots from 10 different plants were surface disinfected with 75% ethanol for 30 s, 3% sodium hypochlorite for 5 min, and then washed three times in sterilize distilled water. The tissues were placed onto Martin Agar (2) at 25°C for 7 days. Nine isolates with morphology similar to Fusarium were obtained from the diseased tissues. Isolates were transferred to potato dextrose agar (PDA) and carnation leaf agar (CLA) and incubated at 25°C. Seven were identified as Fusarium oxysporum and one was F. solani, which have been reported as pathogens of lily in China (1). The other isolate, when grown on PDA, rapidly produced dense, white aerial mycelium that became pink with age and formed red pigments in the medium. On CLA, macroconidia with three to five septate were abundant, relatively slender, and curved to lunate. Microconidia were abundant, oval or pyriform, and one to two celled. Chlamydospores were in chains with smooth exine. The rDNA internal transcribed spacer (ITS) region and a portion of the translation elongation factor 1-alpha (EF-1α) gene of the fungus were amplified, with universal primers ITS1/ITS4 and EF1/EF2 primers respectively (3) and sequenced. In addition, the β-tubulin gene (β-tub) of the fungus was amplified with modified primers Btu-F-F01 (5′-CAGACMGGTCAGTGCGTAA-3′) and Btu-F-R01 (5′-TCTTGGGGTCGAACATCTG-3′) (4). BLASTn analysis showed that the ITS sequences of the isolate (GenBank Accession No. JX989827) had 98.9% similarity with those of F. tricinctum (EF611092, JF776665, and HM776425) and the EF-1α sequences of the isolate (JX989828) had 98.1% similarity with those of F. tricinctum (EU744837 and JX397850). The β-tub sequences of the isolate (JX989829) had 99.0% similarity with those of F. tricinctum (EU490236 and AB587077). The isolate was tested for pathogenicity. Two-month-old ‘Sorbonne’ seedlings were inoculated by placing 5 ml of conidial suspension (about 106 conidia per ml) over the roots of plants in each pot. Control plants were treated with sterile water in the same way. Plants were placed in a greenhouse at 22 to 25°C with a 15-h photoperiod. There were eight plants per pot and three replicates for each treatment. After 3 weeks, 87.5% of the inoculated plants exhibited browning of the root tips, root rot, and yellowing of the leaves, while control plants were symptomless. The pathogen was reisolated from the infected roots and identified as F. tricinctum, thus fulfilling Koch's postulates. To our knowledge, this is the first report of Fusarium wilt of lily caused by F. tricinctum. This information will provide guidance for the control of lily wilt disease and add information useful for the production of lilies. References: (1) C. Li and J. J. Li. Acta Phytopathol. Sin. (in Chinese) 26:192, 1995. (2) J. P. Martin. Soil Sci. 38:215, 1950. (3) K. O'Donnell et al. Proc. Nat. Acad. Sci. U. S. A. 95:2044, 1998. (4) M. Watanabe et al. BMC Evol. Biol. 11:322. 2011.

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First Report of Fusarium stem and root rot of Gerbera jamesonii caused by Fusarium incarnatum in China

Plant Disease ◽

10.1094/pdis-02-21-0440-pdn ◽

2021 ◽

Author(s):

Shuning Chen ◽

Wei Sun ◽

Huizhu Yuan ◽

Xiaojing Yan

Keyword(s):

Root Rot ◽

Vascular System ◽

Elongation Factor ◽

Morphological Observation ◽

Conidial Suspension ◽

Gerbera Jamesonii ◽

Gene Sequences ◽

Plastic Container ◽

First Report ◽

Brown Discoloration

Gerbera (Gerbera jamesonii Bolus) is an important cut flower grown globally. In 2020, gerbera plants (Redaicaoyuan, Baimawangzi, and Hongditan cultivars) with roots, crowns, and stems rot were found in a greenhouse in Nanping, Fujian, China. Approximately 30% of the 60,000 plants showed symptoms. Diseased plants were stunted with chlorotic leaves. The leaves and flower heads were wilted and withered. Brown discoloration with red to black streaks occurred in the vascular system of the crown and stem. The stem pieces (3×3 mm) showing the symptom were surface-disinfected with 1% NaClO for 1 min and washed three times with sterilized water. The stem pieces were then dried and placed on potato dextrose agar (PDA) at 25℃ inside a dark chamber. Ten single-spored isolates were identified as Fusarium incarnatum based on morphological features. White to light brown mycelia were observed among the isolates on PDA medium. Falculate, multicelluar, straight to slightly curved macroconidia produced in monophialide sporodochia without distinctive foot shaped basal cell; and chlamydospores produced in some isolates (Leslie and Summerell). The size of macroconidia was 36.4 ± 5.20 × 4.6 ± 1.3 μm (n = 100) with 3 to 5 septates. Microconidia were mostly 0 to 1 septate measured 14.6 ± 1.9 × 2.6 ± 0.5 μm (n=100). Based on the morphological observation, isolates were further identified by molecular method. The ITS1/4 region combined with partial gene fragments of translation elongation factor (EF-1α, primer EF1/EF2, Geiser et al.) and calmodulin (CAM, primer CL1/CL2A, O’Donnell.) from the isolates were amplified and sequenced. All of the three tested isolates showed identical gene sequences. Sequences amplified from one represented isolate FIN-1 were submitted to Genbank. BLAST searches revealed that ITS1/4 (MW527088), EF-1α (MW556488), and CAM (MW556487) had 99.22%, 99.53%, 99.42% identity compared to F. incarnatum (MN480497, MN233577, and LN901596, respectively) in GenBank. FUSARIUM-ID (Geiser et al. 2004) analysis also showed 99 to 100% similarity with sequences of the F. incarnatum-equiseti species complex (FIESC) (FD_01636 for CAM, FD_01643 for EF-1α). The phylogenetic analysis was conducted using neighbor-joining algorithm based on the ITS, EF-1α, and CAM gene sequences. The isolate was clustered with F. incarnatum clade. Then, the pathogenicity of the fungus was confirmed by performing Koch’s postulates. Pure single-spored cultures were grown on carboxymethyl-cellulose (CMC) medium for sporulation. G. jamesonii plants used for pathogenicity tests were grown on sterilized potting soil in a plastic container to the ten-leaf stage prior to inoculation. Spores harvested from the CMC medium were adjusted to a concentration of 1×105 conidial/ml. Twelve healthy rooted gerbera seedlings were inoculated by drenching 10 ml of the conidial suspension onto roots. Twelve gerbera seedlings treated with 10 ml sterile water served as control treatments. Plants were grown in the glasshouse at temperatures of 23°C, relative humidity >70%, and 16 h light per day. After 10 days, blackening stems and withered leaf edges began to appear on inoculated seedlings, whereas control seedlings remained healthy. F. incarnatum was consistently re-isolated from the symptomatic stems, whereas no isolates were obtained from the control seedlings. The assay was conducted twice. To the best of our knowledge, this is the first report of F. incarnatum causing stem and root rot on G. jamesonii.

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First report of Nigrospora sphaerica causing fruit dried-shrink disease in Akebia trifoliata from China

Plant Disease ◽

10.1094/pdis-11-20-2471-pdn ◽

2021 ◽

Author(s):

Xiujing Hong ◽

Shijia Chen ◽

linchao Wang ◽

Bo Liu ◽

Yuruo Yang ◽

...

Keyword(s):

Elongation Factor ◽

Morphological Characteristics ◽

Its Region ◽

Average Diameter ◽

Ethanol Solution ◽

Likelihood Method ◽

Conidial Suspension ◽

Multiple Sequence ◽

First Report ◽

Pure Cultures

Akebia trifoliata, a recently domesticated horticultural crop, produces delicious fruits containing multiple nutritional metabolites and has been widely used as medicinal herb in China. In June 2020, symptoms of dried-shrink disease were first observed on fruits of A. trifoliata grown in Zhangjiajie, China (110.2°E, 29.4°N) with an incidence about 10%. The infected fruits were shrunken, colored in dark brown, and withered to death (Figure S1A, B). The symptomatic fruits tissues (6 × 6 mm) were excised from three individual plants, surface-disinfested in 1% NaOCl for 30s and 70% ethanol solution for 45s, washed, dried, and plated on potato dextrose agar (PDA) containing 50 mg/L streptomycin sulfate in the dark, and incubated at 25℃ for 3 days. Subsequently, hyphal tips were transferred to PDA to obtain pure cultures. After 7 days, five pure cultures were obtained, including two identical to previously reported Colletotrichum gloeosporioides causing leaf anthracnose in A. trifoliata (Pan et al. 2020) and three unknown isolates (ZJJ-C1-1, ZJJ-C1-2, and ZJJ-C1-3). The mycelia of ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3 were white, and formed colonies of approximate 70 mm (diameter) in size at 25℃ after 7 days on potato sucrose agar (PSA) plates (Figure S1C). After 25 days, conidia were formed, solitary, globose, black, shiny, smooth, and 16-21 μm in size (average diameter = 18.22 ± 1.00 μm, n = 20) (Figure S1D). These morphological characteristics were similar to those of N. sphaerica previously reported (Li et al. 2018). To identify species of ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3, the internal transcribed spacer (ITS) region, β-tubulin (TUB2), and the translation elongation factor 1-alpha (TEF1-α) were amplified using primer pairs including ITS1/ITS4 (Vilgalys and Hester 1990), Bt-2a/Bt-2b (Glass and Donaldson 1995), and EF1-728F/EF-2 (Zhou et al. 2015), respectively. Multiple sequence analyses showed no nucleotide difference was detected among genes tested except ITS that placed three isolates into two groups (Figure S2). BLAST analyses determined that ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3 had 99.73% to N. sphaerica strains LC2705 (KY019479), 100% to LC7294 (KY019397), and 99.79-100% to LC7294 (KX985932) or LC7294 (KX985932) based on sequences of TUB2 (MW252168, MW269660, MW269661), TEF-1α (MW252169, MW269662, MW269663), and ITS (MW250235, MW250236, MW192897), respectively. These indicated three isolates belong to the same species of N. sphaerica. Based on a combined dataset of ITS, TUB2 and TEF-1α sequences, a phylogenetic tree was constructed using Maximum likelihood method through IQ-TREE (Minh et al. 2020) and confirmed that three isolates were N. sphaerica (Figure S2). Further, pathogenicity tests were performed. Briefly, healthy unwounded fruits were surface-disinfected in 0.1% NaOCl for 30s, washed, dried and needling-wounded. Then, three fruits were inoculated with 10 μl of conidial suspension (1 × 106 conidia/ml) derived from three individual isolates, with another three fruits sprayed with 10 μl sterilized water as control. The treated fruits were incubated at 25℃ in 90% humidity. After 15 days, all the three fruits inoculated with conidia displayed typical dried-shrink symptoms as those observed in the farm field (Figure S1E). The decayed tissues with mycelium and spores could be observed on the skin or vertical split of the infected fruits after 15 days’ inoculation (Figure S1F-H). Comparably, in the three control fruits, there were no dried-shrink-related symptoms displayed. The experiment was repeated twice. The re-isolated pathogens were identical to N. sphaerica determined by sequencing the ITS, TUB2 and TEF-1α. Previous reports showed N. sphaerica could cause postharvest rot disease in kiwifruits (Li et al. 2018). To our knowledge, this is the first report of N. sphaerica causing fruits dried-shrink disease in A. trifoliata in China.

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First Report of Sugar Beet Rhizoctonia Crown and Root Rot Caused by Rhizoctonia solani AG-2-2IIIB in Shanxi Province of China

Plant Disease ◽

10.1094/pdis-12-12-1202-pdn ◽

2014 ◽

Vol 98 (3) ◽

pp. 419-419 ◽

Cited By ~ 3

Author(s):

C. Zhao ◽

X. H. Wu

Keyword(s):

Sugar Beet ◽

Rhizoctonia Solani ◽

Root Rot ◽

Its Region ◽

Northern China ◽

Shanxi Province ◽

Molecular Characteristics ◽

First Report ◽

Crown And Root Rot ◽

Wheat Seeds

Sugar beet (Beta vulgaris L.) is grown worldwide as the second largest sugar crop. Sugar beet crown and root rot is an economically serious disease mainly caused by Rhizoctonia solani (teleomorph Thanatephorus cucumeris) AG 2-2 and AG 4 (1). In July 2010, at the 25- to 27-leaf stage, symptoms typically associated with crown and root rot, including dark brown to black lesions at the base of the petioles or circular to oval dark lesions (up to 10.0 mm in diameter) at the taproot, were observed on 15% of sugar beet plants collected from three sites in Shanxi Province, northern China. Pieces of internal root tissues cut from the margins between symptomatic and healthy-appearing tissue were disinfected with 0.5% NaOCl for 2 min, rinsed three times with sterile water, then placed on water ager (WA) for incubation at 25°C in the dark. After 2 days, single hyphal tips of three Rhizoctonia-like isolates (designated SX-RSD1, SX-RSD2, and SX-RSD3) were transferred to potato dextrose ager (PDA). Colonies of all isolates were brown and developed dark brown sclerotia (0.5 to 1.0 mm diameter) on the media surface after 4 and 7 days, respectively. Mycelia were branched at right angles with septa near the branches and slight constrictions at the bases of the branches were present. Average hyphal diameters of the three isolates were 8.1, 7.3, and 7.6 μm, respectively. Hyphal cells were determined to be multinucleate (4 to 9 nuclei per cell) by staining with 4′-6-diamidino-2-phenylindole (DAPI) (2). Anastomosis groups were determined by pairing with reference strains (kindly provided by N. Kondo, Hokkaido University, Japan) (2), and all three isolates anastomosed with R. solani AG-2-2IIIB. All three isolates grew well on PDA at 35°C, which separates AG-2-2IIIB from AG-2-2 IV. The internal transcribed spacer (ITS) region of rDNA was amplified from genomic DNA of these isolates with primers ITS1 (5′-TCCGATGGTGAACCTGCGG-3′)/ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). Sequences (GenBank Accession Nos. KC413984, KC413985, and KC413986) were over 99% identical to those of 19 R. solani AG-2-2 IIIB isolates (e.g., FJ492146.3; strain F510). Therefore, based on the molecular characteristics and the anastomosis assay, these three isolates were identified as R. solani AG-2-2IIIB. To determine the pathogenicity of the isolates, wheat seeds were autoclaved twice for 60 min at 121°C on consecutive days and inoculated with each isolate (3). Subsequently, wheat seeds (three seeds per plant) were placed around 8-week-old sugar beet (cv. HI0305) plants at 2 cm intervals to each root and 10 mm deep in soil. Plants were grown at 25 to 27°C for 7 days in a glasshouse. All inoculated plants developed symptoms of root rot, whereas control plants inoculated with sterilized wheat seeds remained healthy. R. solani AG-2-2IIIB was consistently re-isolated from the symptomatic root tissue and was confirmed by both morphological and molecular characteristics described above, fulfilling Koch's postulates. To our knowledge, this is the first report of R. solani AG-2-2IIIB on sugar beet in Shanxi Province of China. R. solani AG2-2IIIB has been reported to be pathogenic on wheat in China (4), which is often grown in rotation with sugar beet. This rotation could increase the risk of soilborne infection to either crop by R. solani AG2-2IIIB. References: (1) R. M. Harveson et al. Compendium of Beet Diseases and Pests, American Phytopathological Society. St. Paul, MN. 2009. (2) W. C. Kronland and M. E. Stanghellini. Phytopathology. 78:820, 1988. (3) M. J. Lehtonen et al. Plant Pathol. 57:141, 2008. (4) D. Z. Yu et al., Hubei Agric. Sci. 3:39, 2000.

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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 Nectria haematococca Causing Stem Rot of Soybean in China

Plant Disease ◽

10.1094/pdis-07-11-0626 ◽

2012 ◽

Vol 96 (3) ◽

pp. 457-457 ◽

Cited By ~ 1

Author(s):

Y. Gai ◽

R. Pan ◽

D. Xu ◽

M. Deng ◽

W. Chen ◽

...

Keyword(s):

Elongation Factor ◽

Morphological Characteristics ◽

Its Region ◽

Stem Rot ◽

Universal Primers ◽

Conidial Suspension ◽

Fruiting Bodies ◽

Nectria Haematococca ◽

First Report ◽

Soybean Seedlings

In October 2010, soybean (Glycine max) plants growing in commercial soybean fields in Zengcheng City, Guangdong Province developed symptoms consisting of stem and root rot, yellowing, and defoliation of leaves. Reddish, spherical fruiting bodies appeared in lesions that developed on stems. Plants with symptoms were sampled from fields. Fruiting bodies were excised from diseased tissues. Microscopic examination revealed that they were perithecia, globose to pyriform, and measured 197 to 260 μm in diameter and 226 to 358 μm long. When squeezed gently, cylindrical to clavate asci, 7.2 to 9.6 μm in diameter and 75.4 to 92.0 μm long, containing eight ascospores were exuded from the perithecia. Ascospores were ellipsoid to obovate, two celled, slightly constricted at the septum, had longitudinal striations, and measured 4.9 to 6.0 μm in diameter and 10.6 to 15.0 μm long. The fungus was isolated from the basal stem tissues of diseased soybean plants and cultured on potato dextrose agar (PDA) medium amended with streptomycin sulfate. On PDA, the culture developed into blue-pigmented colonies with whitish mycelium that produced oval to cylindrical microconidia. Microconidia had 0 to 1 septum, ranged from 2.5 to 5.2 × 7.6 to 29.4 μm, and were produced on monophialides. Macroconidia were cylindrical to falcate, thick walled, 2 to 5 septa, and 3.5 to 6.0 × 25.4 to 66.8 μm. Chlamydospores were present and ranged from 6.8 to 13.6 × 5.5 to 9.5 μm. Orange-to-reddish perithecia were readily formed in old culture. These morphological characteristics were consistent with descriptions of Nectria haematococca (anamorph Fusarium solani) (1). The rDNA internal transcribed spacer (ITS) region and the fragment of translation elongation factor 1-alpha (EF1-α) genes of the fungus were amplified, respectively, with universal primers ITS1/ITS4 and ef1/ef2 primers and sequenced. BLAST searches showed that the ITS sequences of three isolates (GenBank Accession Nos. JN015069, JN190942, and JN190943) had 99% similarity with those of N. haematococca(GenBank Accession Nos. DQ535186, DQ535185, and DQ535183) and the EF1-α sequences of three isolates (GenBank Accession Nos. JN874641, JN874642, and JN874643) had 100% similarity with those of F. solani (GenBank Accession Nos. DQ247265 and DQ247327). Completion of Koch's postulates confirmed the pathogenicity of the isolates in a replicated experiment. Thirty-day-old soybean seedlings of cultivar Huaxia No. 3 were inoculated by soaking their root systems in a conidial suspension (106 conidia per ml) for 30 min and then transplanted in plastic pots (20 cm in diameter) and incubated at 25 ± 2°C in a greenhouse. Control plants were treated with sterile water in the same way. There were four plants per pot and there were six replicates for each treatment. Within 3 weeks, more than 70% of the inoculated plants exhibited symptoms of leaf yellowing, stem rot, and root rots while control plants were symptomless. N. haematococca was reisolated from the diseased plants. To our knowledge, this is the first report of N. haematococca causing stem rot of soybean in China and the first description of sexual reproduction of F. solani causing soybean stem rot in nature. This pathogen may pose a serious threat to soybean production in China where soybean is a main crop. Reference: (1) C. Booth. The Genus Fusarium. CAB International, Wallingford, UK, 1971.

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First report of root rot caused by Fusarium armeniacum on American ginseng in China

Plant Disease ◽

10.1094/pdis-12-19-2554-pdn ◽

2020 ◽

Author(s):

Ximei Zhang ◽

Yanmeng Bi ◽

Junfei LI ◽

Huihui SHAO ◽

XIAO LIN JIAO ◽

...

Keyword(s):

Root Rot ◽

Apical Cell ◽

Elongation Factor ◽

American Ginseng ◽

Ginseng Root ◽

Conidial Suspension ◽

First Report ◽

Tubulin Genes ◽

One Year ◽

Whole Plants

American ginseng (Panax quinquefolius) is an important medicinal plant cultivated in China since the 1980s. Its dried roots are used for food, health care products, and medicine in China (Yuan et al. 2010). Root rot caused by Fusarium spp. was a major disease, with 33 to 41% incidence surveyed in main production areas of Wendeng County (121.80 °E, 37.09 °N) in Shandong Province, China in 2016 to 2019. Symptoms included soft, water-soaked, dark brown to black lesions on the roots. Lesions progressed and the inner parts gradually disintegrated. One-year-old diseased roots were collected in September 2016. Symptomatic tissues were surface-sterilized in 75% ethanol for 30 s and 0.8% NaOCl for 3 min, rinsed in sterile water, plated on potato dextrose agar (PDA), and incubated at 25°C in darkness. Single colonies were then obtained and transferred to carnation leaf agar (CLA) (Burgess et al. 1993) for growth at 25°C with a 12-h photoperiod. Colonies cultured on PDA for 7 days were white to light pink, turning to apricot pigmentation in color. After 30 days on CLA, the colonies produced elongate, falcate macroconidia having 3 to 5 septa, with a long, tapering and curved apical cell, and having the size ranging from 31.1 to 45.6 μm long x 4 to 4.6 μm wide. Microconidia were zero to 1septate, ellipsoid to ovoid and varied in size from 9.5 to 16.8 μm long x 3 to 3.2 μm wide. Chlamydospores formed abundantly, in chains or clusters. This fungus was identified as F. armeniacum (Burgess et al. 1993). Identification was confirmed by sequencing three DNA regions including the internal spacer ribosomal DNA (ITS), elongation factor 1α and β-tubulin genes (Lu et al. 2019). The three DNA regions (MN417271, MG457199, and MN427653) had 100% homology to the sequences of F. armeniacum (KJ737378, HM744664 and HQ141640) (Wang et al. 2015, Yli-Mattila et al. 2011). Pathogenicity tests were conducted on 1- to 2-year-old bare roots and 2-year-old whole plants. For root inoculation, 14 healthy roots were inoculated with two mycelial PDA plugs/root. After 3 to 10 days at 25°C, all the inoculated roots showed water-soaked and root rot symptoms while no lesions were observed in the control roots. For plant inoculation, eight seedlings planted in pots filled with sterilized soil were inoculated by pouring a conidial suspension of 1×105 conidia/ml at 30 ml/pot. Eight seedlings inoculated with sterilized water served as the controls. After 90 days, only 37.5% of the roots survived with typical root rot symptoms whereas the control plants remained symptomless. F. armeniacum was re-isolated from symptomatic roots but not from the control roots. Besides F. armeniacum, F. solani and F. oxysporum that have been reported to be associated with American ginseng root rot in China and Canada (Reeleder et al. 2002; Punja et al. 2008) were also obtained from the diseased root samples in this study. However, the development of root rot caused by F. armeniacum was much more rapid and its symptoms were more severe. Moreover, F. armeniacum could directly infect American ginseng with no wound requirement. F. armeniacum was previously reported on Glycine max (Leguminosae) (Ellis et al. 2012), Platycodon grandiflorus (Campanulaceae) (Wang et al. 2015) and natural grasses (Poaceae) (Nichea et al. 2015). This is the first report of F. armeniacum causing root rot on American ginseng in China. As this species is more virulent to American ginseng, more research is needed to work on this disease.

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First report of blight on Fritillaria thunbergii caused by Fusarium oxysporum in Zhejiang Province, China

Plant Disease ◽

10.1094/pdis-11-21-2407-pdn ◽

2021 ◽

Author(s):

Yiwen Xu ◽

Zhenyan Cao ◽

Yihua Yang ◽

Xuping Shentu ◽

Xiaoping Yu

Keyword(s):

Early Stage ◽

Science And Technology ◽

Elongation Factor ◽

Morphological Characteristics ◽

Its Region ◽

Zhejiang Province ◽

Gastric Ulcers ◽

Conidial Suspension ◽

Sterile Water ◽

First Report

Fritillaria thunbergii Miq. (Zhe beimu), is an oldest known homeopathic traditional folk medicinal plant in Zhejiang Province, China. The bulbs are medicinally important curing cough, inflammation, gastric ulcers, hypertension, diarrhea, and bronchitis (Nile et al. 2021). In April of 2018 and 2019, symptoms of blight on F. thunbergii were observed with an incidence of 20–25% in Cixi city, Zhejiang Province. At the early stage of this disease, the stalk turned brown, then the whole bulbs rotted within a few days. Symptomatic bulbs were cut into small pieces (1.0 cm × 1.0 cm) and disinfected successively by submersion in 75% ethanol for 30 seconds and 1% NaClO for 30 seconds under aseptic conditions. After rinsing with sterile water three times and air drying, segments were placed on potato dextrose agar (PDA). After incubation at 28 ℃ for 7 days in the dark, the hyphae were observed white fluffy, spreading from the middle to the whole plate. Macroconidia were falciform with zero to four septa and (11.0-39.0) × (3.0-5.0) μm in size. Microconidia were fusiform with zero to two septa (4.0-7.0) × (2.6-3.0) μm in size. Based on morphological characteristics of macroconidia, and microconidia, isolates were identified as F. oxysporum (Lombard, L. et al., 2019). The internal transcribed spacer (ITS) region, translation elongation factor (EF-1α), and RNA polymerase second largest subunit (RPB2) gene were amplified and sequenced respectively using ITS1/ITS4, EF1/EF2 and 5f2/7cr primers (O’Donnell et al., 2010). BLASTN analysis of FUSARIUM-ID using ITS (Accession NO.MZ268594), EF-1α (Accession NO.MZ292517) and RPB2(Accession NO.MZ292516) showed 95.2%, 100%, and 99.11% identity to F. oxysporum species complex isolates NRRL43730, NRRL38599 and NRRL38302, respectively. Based on the morphological and molecular characters, the pathogen was identified as F. oxysporum. To verify pathogenicity, ten healthy F. thunbergii plants were used for inoculation tests. One milliliter of a conidial suspension (106 conidia ml-1) was pipetted onto the soil around the base of F. thunbergii plants per pot. Ten plants, which were treated with sterile water, were used as the control. All plants were maintained in a climatic chamber (26 ± 1 ℃, 70–80% relative humidity and a photoperiod of 16:8 [L: D] h). Seven days later, all inoculated plants showed typical symptoms of blight identical to those observed in the fields. Control plants remained symptomless and healthy. The pathogenicity analysis was repeated three times. Pathogens re-isolated from symptomatic plants were identified as F. oxysporum by morphology observation and sequence analysis. To our knowledge, this is the first report of blight caused by F. oxysporum on F. thunbergii in Zhejiang Province, China. Acknowledgments: The author(s) declare no conflict of interest. Funding: This work was supported by Zhejiang Provincial Program for Science and Technology Development (2017C32006, 2018C02030) and the Student Science and Technology Innovation Project of China Jiliang University (2021YW95). References: Nile et al. 2021.J. Food and chemical toxicology, 153:112289. Hami, A. et al., 2021. J. Scientific Reports. 3610.11.1. Lombard, L. et al., 2019. PERSOONIA, 43:1-47. O’Donnell, K., et al. 2010. J. Clin. Microbiol. 48: 3708-3718.

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