First Report of Lasiodiplodia theobromae Causing Branch Dieback on Castor Bean in Zhanjiang, China

Castor bean (Ricinus communis L.) is an oil crop of significant economic importance in the industry and medicine. In August 2019, a branch dieback disease was observed on castor bean in a field in Zhanjiang (21.17°N, 110.18°E), China. The incidence rate was 35% (n=600 investigated plants). Symptoms were discoloration of leaves, branch dieback, and discoloration of internal stem tissues. The disease had spread to the whole branches and causing the plant to die. Seven diseased branches were collected from seven plants. Margins between healthy and diseased tissues were cut into 2 mm × 2 mm pieces. The surfaces were disinfested with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Then, the samples were rinsed thrice in sterile water, placed on PDA, and incubated at 28 °C. Pure cultures were obtained by transferring the hyphal tips to new PDA plates. Eighteen isolates were obtained (the isolate rate of 75%), which were the same fungus on the basis of morphological characteristics and molecular analysis of the internal transcribed spacer (ITS). A single representative isolate (RiB-1) was used for further study. The colony of RiB-1 was 5 cm in diameter on the 5th day on the PDA culture. The colony was greenish gray with an irregularly distributed and fluffy aerial mycelium, which turned black after 10 days. The mature conidia were 21.3–26.5 µm × 12.2–15.7 µm in size (n=100) and had two ovoid, dark brown cells with longitudinal striations. The morphological characteristics of the colonies were consistent with the description of Lasiodiplodia sp. (Alves et al. 2008). Three regions of the ITS, translation elongation factor (EF1-α), and β-tubulin genes were amplified and sequenced with the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Alves et al. 2008), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The resulting sequences were deposited in the GenBank under accession numbers MN759432 (ITS), MN719125 (EF1-α), and MN719128 (β-tubulin). BLASTn analysis demonstrated that these sequences were 100% identical to the corresponding ITS (MK530052), EF1-α (MK423878), and β-tubulin (MN172230) sequences of L. theobromae. Based on the morphological and molecular data, RiB-1 was determined as L. theobromae. A pathogenicity test was performed in a greenhouse with 80% relative humidity at 25 °C to 30 °C. Ten healthy plants of Zi Bi No. 5 castor bean (1-month-old) were grown in pots with one plant in each pot. Five pots were wound-inoculated with 5-mm-diameter mycelial plugs obtained from 7-day cultures. Five additional pots treated with PDA plugs served as the controls. Inoculated stems were moisturized with sterile cotton for five days. The test was conducted three times. Disease symptoms, similar to those in the field, were observed on the inoculated plants two weeks after inoculation, and L. theobromae was 100% reisolated from the inoculated plants. The control plants remained symptomless, and reisolations were unsuccessful. These results consistent with Koch’s postulates. L. theobromae (Lima et al. 1997) and L. hormozganensis (Fábio et al. 2018) had been reported to cause stem rot on castor bean in Brazil, but whether L. theobromae caused the branch dieback on castor bean in China has not been reported yet. Thus, this study is the first report of L. theobromae causing the branch dieback on castor bean in Zhanjiang, China. This study provides an important reference for the control of the disease.

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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 Diaporthe ueckerae causing stem canker on soybean (Glycine max L.) in Colombia

Plant Disease ◽

10.1094/pdis-04-21-0718-pdn ◽

2021 ◽

Author(s):

Nathali López-Cardona ◽

YUDY ALEJANDRA GUEVARA ◽

Lederson Gañán-Betancur ◽

Carol Viviana Amaya Gomez

Keyword(s):

Management Strategies ◽

Elongation Factor ◽

Morphological Characteristics ◽

Aerial Mycelium ◽

Stem Canker ◽

Growth Stages ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Glycine Max L ◽

Soybean Plants

In October 2018, soybean plants displaying elongated black to reddish-brown lesions on stems were observed in a field planted to the cv. BRS Serena in the locality of Puerto López (Meta, Colombia), with 20% incidence of diseased plants. Symptomatic stems were collected from five plants, and small pieces (∼5 mm2) were surface sterilized, plated on potato dextrose agar (PDA) and incubated for 2 weeks at 25°C in darkness. Three fungal isolates with similar morphology were obtained, i.e., by subculturing single hyphal tips, and their colonies on PDA were grayish-white, fluffy, with aerial mycelium, dark colored substrate mycelium, and produced circular black stroma. Pycnidia were globose, black, occurred as clusters, embedded in tissue, erumpent at maturity, with an elongated neck, and often had yellowish conidial cirrus extruding from the ostiole. Alpha conidia were observed for all isolates after 30 days growth on sterile soybean stem pieces (5 cm) on water agar, under 25ºC and 12 h light/12h darkness photoperiod. Alpha conidia (n = 50) measured 6.0 – 7.0 µm (6.4 ± 0.4 µm) × 2.0 – 3.0 µm (2.5± 0.4 µm), were aseptate, hyaline, smooth, ellipsoidal, often biguttulate, with subtruncate base. Beta conidia were not observed. Observed morphological characteristics of these isolates were similar to those reported in Diaporthe spp. by Udayanga et al. (2015). DNA from each fungal isolate was used to sequence the internal transcribed spacer region (ITS), and the translation elongation factor 1-α (TEF1) gene, using the primer pairs ITS5/ITS4 (White et al. 1990) and EF1-728F/EF1- 986R (Carbone & Kohn, 1999), respectively. Results from an NCBI-BLASTn, revealed that the ITS sequences of the three isolates (GenBank accessions MW566593 to MW566595) had 98% (581/584 bp) identity with D. miriciae strain BRIP 54736j (NR_147535.1), whereas the TEF1 sequences (GenBank accessions MW597410 to MW597412) had 97 to 100% (330-339/339 bp) identity with D. ueckerae strain FAU656 (KJ590747). The species Diaporthe miriciae R.G. Shivas, S.M. Thomps. & Y.P. Tan, and Diaporthe ueckerae Udayanga & Castl. are synonymous, with the latter taking the nomenclature priority (Gao et al. 2016). According to a multilocus phylogenetic analysis, by maximum likelihood, the three isolates clustered together in a clade with reference type strains of D. ueckerae (Udayanga et al. 2015). Soybean plants cv. BRS Serena (growth stages V3 to V4) were used to verify the pathogenicity of each isolate using a toothpick inoculation method (Mena et al. 2020). A single toothpick colonized by D. ueckerae was inserted directly into the stem of each plant (10 plants per isolate) approximately 1 cm below the first trifoliate node. Noncolonized sterile toothpicks, inserted in 10 soybean plants served as the non-inoculated control. Plants were arbitrarily distributed inside a glasshouse, and incubated at high relative humidity (>90% HR). After 15 days, inoculated plants showed elongated reddish-brown necrosis at the inoculated sites, that were similar to symptoms observed in the field. Non-inoculated control plants were asymptomatic. Fungal cultures recovered from symptomatic stems were morphologically identical to the original isolates. This is the first report of soybean stem canker caused by D. ueckerae in Colombia. Due to the economic importance of this disease elsewhere (Backman et al. 1985; Mena et al. 2020), further research on disease management strategies to mitigate potential crop losses is warranted.

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First Report of Botryosphaeria dothidea, Diplodia seriata, and Neofusicoccum luteum Associated with Canker and Dieback of Grapevines in Tunisia

Plant Disease ◽

10.1094/pdis-05-13-0530-pdn ◽

2014 ◽

Vol 98 (3) ◽

pp. 420-420 ◽

Cited By ~ 6

Author(s):

S. Chebil ◽

R. Fersi ◽

A. Yakoub ◽

S. Chenenaoui ◽

M. Chattaoui ◽

...

Keyword(s):

Morphological Characteristics ◽

Aerial Mycelium ◽

Its2 Region ◽

Pathogenicity Test ◽

Botryosphaeria Dothidea ◽

Diplodia Seriata ◽

First Report ◽

Grapevine Dieback ◽

Pinus Spp ◽

Neofusicoccum Luteum

In 2011, common symptoms of grapevine dieback were frequently observed in 2- to 5-year-old table grape (Vitis vinifera L.) cvs. in four vineyards located in northern Tunisia. The symptoms included dead spur and cordons, shoot dieback, and sunken necrotic bark lesions, which progressed into the trunk resulting in the death of large sections of the vine. Longitudinal and transversal sections of cordons and spurs from symptomatic vines revealed brown wedge-shaped cankers of hard consistency. Twelve symptomatic samples from spur and cordons were collected, surface disinfected by dipping into 5% (v/v) sodium hypochlorite for 2 min, and small pieces from the edge of necrotic and healthy tissue were removed and plated onto potato dextrose agar (PDA) at 25°C in the dark. Based on colony and conidia morphological characteristics, isolates were divided in three species, named Diplodia seriata, Botryosphaeria dothidea, and Neofusicoccum luteum. D. seriata colonies were gray-brown with dense aerial mycelium producing brown cylindric to ellipsoid conidia rounded at both ends and averaged 22.4 × 11.7 μm (n = 50). B. dothidea colonies were initially white with abundant aerial mycelium, gradually becoming dark green olivaceous. Conidia were fusiform to fusiform elliptical with a subobtuse apex and averaged 24.8 × 4.7 μm (n = 50). N. luteum colonies were initially pale to colorless, gradually darkening with age and becoming gray to dark gray producing a yellow pigment that diffuses into the agar. Conidia were hyaline, thin-walled, aseptate, fusiform to fusiform elliptical, and averaged 19.8 × 5.5 μm (n = 50). Identity of the different taxa was confirmed by sequence analyses of the internal transcribed spacer (ITS1-5.8S-ITS2) region of the rDNA and part of the elongation factor 1-alpha (EF1-α) gene. BLAST analysis of sequences indicated that six isolates were identified as D. seriata (GenBank: AY259094, AY343353), one isolate as B. dothidea (AY236949, AY786319) and one isolate as N. luteum (AY259091, AY573217). Sequences were deposited in GenBank under accessions from KC178817 to KC178824 and from KF546829 to KF546836 for ITS region and EF1-α gene, respectively. A pathogenicity test was conducted on detached green shoots cv. Italia for the eight Botryosphaeriaceae isolates. Shoots were inoculated by placing a colonized agar plug (5 mm diameter) from the margin of a 7-day-old colony on fresh wound sites made with a sterilized scalpel. Each wound was covered with moisturized cotton and sealed with Parafilm. Control shoots were inoculated using non-colonized PDA plugs. After 6 weeks, discoloration of xylem and phloem and necrosis with average length of 38.8, 17.6, and 11.2 mm were observed from inoculated shoots with D. seriata, N. luteum, and B. dothidea, respectively, and all three fungi were re-isolated from necrotic tissue, satisfying Koch's postulates. Control shoots showed no symptoms of the disease and no fungus was re-isolated. In Tunisia, Botryosphaeria-related dieback was reported only on citrus tree caused by B. ribis (2), on Pinus spp. caused by D. pinea (4), on Quercus spp. caused by D. corticola (3), and on olive tree (Olea europea) caused by D. seriata (1). To our knowledge, this is the first report of D. seriata, B. dothidea, and N. luteum associated with grapevine dieback in Tunisia. References: (1) M. Chattaoui et al. Plant Dis. 96:905, 2012. (2) H. S. Fawcett. Calif. Citrogr. 16:208, 1931. (3) B. T. Linaldeddu et al. J. Plant Pathol. 91:234. 2009. (4) B. T. Linaldeddu et al. Phytopathol. Mediterr. 47:258, 2008.

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First Report of Lasiodiplodia theobromae Associated with Decline of Grapevine Rootstock Mother Plants in Spain

Plant Disease ◽

10.1094/pdis-92-5-0832b ◽

2008 ◽

Vol 92 (5) ◽

pp. 832-832 ◽

Cited By ~ 13

Author(s):

A. Aroca ◽

R. Raposo ◽

D. Gramaje ◽

J. Armengol ◽

S. Martos ◽

...

Keyword(s):

Elongation Factor ◽

Aerial Mycelium ◽

Its Region ◽

Pathogenic Fungi ◽

Vascular Lesions ◽

Malt Extract ◽

Pathogenicity Test ◽

Lasiodiplodia Theobromae ◽

First Report ◽

Mother Plants

A field of Richter 110 rootstock mother plants in Valencia Province (eastern Spain) was surveyed during November 2006 to study the mycoflora of declining plants. Two canes with stunted leaves were collected from a plant with a reduced number of shoots. No cankers or vascular lesions were observed in the collected canes. Six wood chips (1 to 2 mm thick) were taken from one basal fragment (3 to 4 cm long) of each cane, surface sterilized in 70% ethanol for 1 min, and plated on malt extract agar supplemented with 0.5 g L–1 of streptomycin sulfate. Petri dishes were incubated for 7 days at 25°C. A fungus was consistently isolated from all samples that showed the following characteristics: colonies grown on potato dextrose agar (PDA) at 25°C developed a white, aerial mycelium that turned gray after 4 to 6 days and produced pycnidia after 1 month on sterile grapevine slivers of twigs placed on the PDA surface; conidia from culture were ellipsoidal, thick walled, initially hyaline, nonseptate, and measuring 20 to 25 (22.5) × 12 to 14 (13) μm; aged conidia were brown, 1-septate with longitudinal striations in the wall; and pseudoparaphyses variable in form and length were interspersed within the fertile tissue. The fungus was identified as Lasiodiplodia theobromae (Pat.) Griffon & Maubl. from the above characteristics (2). Identity was confirmed by analysis of the nucleotide sequences of the internal transcribed spacer (ITS) region from the rRNA repeat and part of the translation elongation factor 1-alpha (EF1-α) and the β-tubulin (B-tub) genes, as done elsewhere (1,3). BLAST searches at GenBank showed a high identity with reference sequences (ITS: 100%, EF1-α: 97%; B-tub: 99%). Representative sequences of the studied DNA regions were deposited at GenBank (Accession Nos.: ITS: EU254718; EF1-α: EU254719; and B-tub: EU254720). A pathogenicity test was conducted on 1-year-old grapevine plants cv. Macabeo grafted onto Richter 110 rootstocks maintained in a greenhouse. A superficial wound was made on the bark of 10 plants with a sterilized scalpel, ≈10 cm above the graft union. A mycelial plug obtained from the margin of an actively growing fungal colony (isolate JL664) was placed in the wound and the wound was wrapped with Parafilm. Ten additional control plants were inoculated with sterile PDA plugs. All control plants grew normally, and the inoculation wound healed 3 months after inoculation. Plants inoculated with L. theobromae showed no foliar symptoms in the same period, but developed cankers variable in size surrounding the inoculation sites. Vascular necroses measuring 8.4 ± 1.5 cm (mean ± standard error) developed in the inoculated plants that were significantly longer than the controls (0.3 ± 0.2 cm). The pathogen was reisolated from all inoculated plants and no fungus was reisolated from the controls. These results confirmed the pathogenicity of L. theobromae to grapevine and points to a possible involvement of L. theobromae in the aetiology of grapevine decline as previously reported (3,4). To our knowledge, this is the first report of L. theobromae isolated from grapevine in Spain. References: (1) J. Luque et al. Mycologia 97:1111, 2005. (2) E. Punithalingam. No. 519 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1976. (3) J. R. Úrbez-Torres et al. Plant Dis. 90:1490, 2006. (4) J. M. van Niekerk et al. Phytopathol. Mediterr. 45(suppl.):S43, 2006.

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First Report of Fusarium Maize Ear Rot Caused by Fusarium meridionale in China

Plant Disease ◽

10.1094/pdis-01-14-0108-pdn ◽

2014 ◽

Vol 98 (8) ◽

pp. 1156-1156 ◽

Cited By ~ 4

Author(s):

H. Zhang ◽

W. Luo ◽

Y. Pan ◽

J. Xu ◽

J. S. Xu ◽

...

Keyword(s):

Fusarium Species ◽

Elongation Factor ◽

Morphological Characteristics ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Fusarium Ear Rot ◽

Ear Rot ◽

Translation Elongation Factor ◽

Translation Elongation ◽

First Report

Fusarium is an important genus of fungal pathogens that are responsible for devastating diseases, such as Fusarium ear rot on maize, which may result in yield losses and/or mycotoxin contamination. In September 2013, a survey to determine population composition of Fusarium species on maize was conducted at 22 fields in 18 counties in Gansu Province. Maize ears with clear symptoms (with a white to pink- or salmon-colored mold at the ear tip) were collected. Symptomatic seeds were surface-sterilized with 70% ethanol and 10% sodium hypochlorite and rinsed three times with sterile water to eliminate hypochlorite residues. After drying on sterile filter paper, the seeds were placed on potato dextrose agar (PDA) and incubated at 25°C in the dark for 3 days. Mycelium that was characteristic of Fusarium spp. (2) was purified by transferring single spores to fresh PDA. Fusarium species were identified by morphological characteristics (2), multilocus genotyping assay (MLGT) (3), and sequence analysis of the translation elongation factor-1α (TEF) gene. Several Fusarium species were identified and Fusarium verticillioides and F. proliferatum were the predominant species. Based on MLGT, two strains from Chenghong County were identified as F. meridionale with NIV chemotype, a species in F. graminearum species complex (FGSC). Morphological characteristics were also identical to FGSC. Colonies grew rapidly on PDA and produce relatively large amounts of dense mycelia and red pigments. Slender, thick-walled, and moderately curved or straight macroconidia were observed with 5- to 6-septate. Furthermore, conidia on SNA also showed typical characteristics of F. meridionale, as the dorsal and ventral lines were often parallel and gradually curved. Sequences comparison of the partial translation elongation factor (TEF-1α, 644 bp) gene (1) was used to validate these observations. BLASTn analysis with the FUSARIUM-ID database revealed 100% sequence identity to F. meridionale (GenBank Accession No. KJ137017). Thus, both morphological and molecular criteria supported identification of the strains as F. meridionale. A pathogenicity test was performed on Zhengdan958, the maize variety with the largest planted acreage in China. Four days after silk emergence, 2 ml conidial suspension (105 macroconidia/ml) of each isolate were injected into each of 10 maize ears through silk channel. Control plants were inoculated with sterile distilled water. Typical FER symptoms (reddish-white mold) was observed on inoculated ears and no symptoms were observed on water controls. Koch's postulates were fulfilled by re-isolating the same fungus from the infected seeds. F. meridionale was one of the pathogens causing Fusarium head blight on wheat and barley in China and produced nivalenol (4,5) and it also has been isolated from maize in Korea and Nepal. To our knowledge, this is the first report of F. meridionale causing Fusarium ear rot on maize in China. Further studies on biological characteristics such as temperature sensibility and fungicide resistance are needed to gain a better understanding of this new pathogen. References: (1) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (3) T. J. Ward et al. Fungal Genet. Biol. 45:473, 2008. (4) L. Yang et al. Phytopathology 98:719, 2008. (5) H. Zhang et al. Plos one 7:e31722, 2012.

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First Report of Vascular Wilt Caused by Fusarium redolens on Lentil in Italy

Plant Disease ◽

10.1094/pdis-92-7-1132c ◽

2008 ◽

Vol 92 (7) ◽

pp. 1132-1132 ◽

Cited By ~ 5

Author(s):

L. Riccioni ◽

A. Haegi ◽

M. Valvassori

Keyword(s):

Vascular Tissue ◽

Fusarium Species ◽

Elongation Factor ◽

Morphological Characteristics ◽

Its Region ◽

Its Sequences ◽

Homology Search ◽

Control Treatment ◽

Pathogenicity Test ◽

First Report

Lentil (Lens culinaris Medik.) is a traditional crop in Sicily, Italy. Near Villalba (Caltanissetta), a local lentil landrace, “Lenticchia di Villalba”, is commonly grown. From 2002 to 2004, wilt was observed in five lentil fields (≈1 ha each) at rates from 5 to 20%. Affected plants were yellow and stunted with discoloration in the vascular tissue of stems and crowns. Pieces of brown vascular tissue from stems were disinfested in 2% sodium hypochlorite for 2 min, rinsed with sterile distilled water, placed on potato dextrose agar, and incubated at 23°C. Isolates with morphological characteristics of Fusarium oxysporum Schlecht.:Fr. (2) were consistently recovered from affected plants. For molecular identification of five isolates, the rDNA internal transcribed spacer (ITS) region and a portion of the elongation factor EF-1α were sequenced using ITS5/4 and EF1/2 primers, respectively (1). Two sequences of the ITS region were obtained: a 468-bp sequence from isolates ER1259, ER1260, and ER1275 (submitted as GenBank Accession No. EU159118) and a 483-bp sequence from isolates ER1274 and ER1276 (submitted as GenBank Accession No. EU281661). The two sequences shared 93% similarity. A sequence homology search using the NCBI BLAST program revealed that the first sequence had 100% homology with the ITS sequences of more than 50 F. oxysporum isolates of various formae speciales in GenBank and the second shared 100% homology with the ITS sequences of five isolates of F. redolens Wollenw. (e.g., GenBank Accession No. X94169 of the strain CBS 360.87). Amplification of the EF-1α produced a sequence from isolates ER1274 and ER1276 (submitted as GenBank Accession No. EU281660) with 99 to 100% homology to sequences of F. redolens and a sequence from strains ER1259, ER1275, and ER1260 (submitted as GenBank Accession No. EU281659) with 100% homology to that of more than 50 F. oxysporum strains in GenBank. Although F. redolens and F. oxysporum are morphologically similar, recent molecular studies have shown that they are distinct and phylogenetically distant species (3). On the basis of genetic sequences, isolates ER1274 and ER1276 were identified as F. redolens. These isolates were evaluated for pathogenicity on lentil. For each isolate, 10 2-week-old seedlings of “Lenticchia di Villalba” were inoculated by submerging roots in a suspension of 2.5 × 106 conidia/ml for 10 min. Plants were put into separate tubes containing 70 ml of a nutritional liquid medium (7 ml of HydroPlus Olikani per liter; Yara, Nanterre, France) and incubated in a growth chamber at 20°C with 12 h of light per day. Seedlings dipped in sterile water served as the control treatment. The pathogenicity test was repeated twice. Inoculated seedlings started to wilt 1 week after inoculation and developed root rot and vascular discoloration. After 2 weeks, 70% of the inoculated plants were affected by both isolates and 40 and 10% died when inoculated with ER1274 and ER1276 isolates, respectively. F. redolens was consistently reisolated from the stems of wilted plants. Noninoculated plants remained healthy. Currently, only F. oxysporum f. sp. lentis Vasud. and Sriniv. has been reported as the cause of Fusarium wilt of lentil. To our knowledge, this is the first report of F. redolens as a pathogen on lentil. References: (1) R. P. Baayen et al. Phytopathology 91:1037, 2001. (2) P. E. Nelson et al. Fusarium Species: An Illustrated Manual for Identification. The Pennsylvania State University Press, University Park, 1983. (3) K. O'Donnell et al. Mycologia 90:465, 1998.

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First report of Fusarium lateritium causing shoots dieback of Acer negundo in Europe

Plant Disease ◽

10.1094/pdis-06-21-1294-pdn ◽

2021 ◽

Author(s):

Katarzyna Patejuk ◽

Anna Baturo-Ciesniewska ◽

Kamil Najberek ◽

Wojciech Pusz

Keyword(s):

Invasive Plant ◽

Aerial Mycelium ◽

Enemy Release ◽

Morphological Identification ◽

Pathogenicity Test ◽

First Report ◽

Acer Negundo ◽

Freeze Dried ◽

Wide Range ◽

Β Tubulin

Box elder (Acer negundo) is a tree native to North America. In Europe it is considered a dangerous invasive species, and assigned to the highest (4th) category of environmental hazard (Tokarska-Guzik et al. 2012). The tree can threaten a wide range of ecosystems and compete with the native flora. The shoot dieback was observed on 20% of boxelder in July 2018 and 2019 in Bryzgiel (N53°59.963' E23°04.324') in NE Poland (Europe). Young trees (10-15 yr. old) with visible symptoms were observed in a small group on the rural roadside. Infected shoots were chlorotic. There were visible shallow cracks on the bark and brown discoloration in sapwood inside infected branches. Symptomatic shoots were collected in sterile envelopes, surface disinfected with 95% ethanol. Twelve fragments of wood were cut from the border of living and dead tissue, and then divided into 3-5 mm pieces, placed on PDA medium and incubated at 21°C. After 10 days ten Fusarium spp. strains were obtained. Pure cultures were derived by monosporic isolation. The identification of the isolates was initially based on morphology and molecular genotyping (Leslie & Summerell 2006). On PDA, strains produced white, dense, floccose aerial mycelium with a pink surface. The underside of the petri dish was brown. Growth of the colony was relatively slow and reached Ø 3.5 cm after two weeks. Microscopic observation revealed the presence of macroconidia located in a few orange sporodochia. Macroconidia were slightly curved, with dimensions of 38-45 µm × 3.2-3,5 µm, 4-5 septate, with well-formed foot cell and beak on the apex. On aerial hyphae, single intercalary chlamydospores were present. Microconidia were not found. Morphological identification was confirmed by sequencing the ITS regions, the TEF-1α and β-tubulin genes for representative isolates. Mycelia were grown on PDB and freeze dried prior to genomic DNA extraction using the CTAB method. Sequences of two isolates were deposited in GenBank as MN186748 and MN588156 for ITS; MZ191070 and MZ191072 for TEF-1α; and MZ191069 and MZ191071 for TEF-1α. BLASTn search in the NCBI database revealed 100, 98 and >99% similarities of ITS, TEF-1α and β-tubulin with F. lateritium isolates LC171689, KT350607 and FN554618 respectively. A pathogenicity test was conducted on five first year Ø 0.6-0.8cm shoots from a 10-year-old tree. Before inoculation their surface was disinfected with 95% ethanol. Then, bark of the twigs was split longitudinally with a sterile blade and pieces of 10-day-old aerial mycelium grown on PDA were applied on the wound sites. Control samples were inoculated with sterile distilled water only. Inoculated areas were covered with parafilm. First sign of infection was observed after three weeks, as a dark lesion in the place of inoculation and chlorosis. Three weeks later the brown ring on the sapwood was marked in the shoot cross-section. Morphologically identical to the original, F. lateritium isolate was reisolated from the infected tissues, thus fulfilling Koch’s postulates. F. lateritium is a species closely associated with trees and shrubs (Leslie & Summerell 2006). However, it has not been recorded on boxelder and this is the first report of F. lateritium causing dieback of boxelder maple. According to the Enemy Release Hypothesis (Elton 1958), new pathogens appearing on alien species can be an indicator of developing environmental resistance to the outlander, which indicate the grade of their domestication. This kind of notification poses a crucial role in invasion monitoring and the search for new biocontrol methods of invasive plant species.

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First Report of Branch Blight of Tree Peony Caused by Phoma glomerata in China

Plant Disease ◽

10.1094/pdis-12-12-1129-pdn ◽

2013 ◽

Vol 97 (8) ◽

pp. 1114-1114 ◽

Cited By ~ 4

Author(s):

D. Zhao ◽

Y. B. Kang

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

Aerial Mycelium ◽

Its2 Region ◽

Tree Peony ◽

Disease Symptoms ◽

First Report ◽

Plastic Bag ◽

Pure Cultures ◽

Branch Dieback

Tree peony (Paeonia suffruticosa Andrews) is a perennial woody deciduous shrub native to China and famous for its beautiful flowers. Starting in early autumn 2010, blighted branches of tree peony were detected in the International Peony Garden in Luoyang. The disease incidence was greater than 10% and disease symptoms included bulb atrophy and twig and branch dieback. Pycnidia were embedded within the bark of diseased branches. They were small, black, ostiolate, and measured 145 to 275 × 140 to 251 μm. Pycnoconidia were single-celled, hyaline or sandy beige, rounded to ellipsoidal, and 3.9 to 10.3 × 2.3 to 7.0 μm. Pure cultures were obtained by plating the pycnoconidia on potato dextrose agar (PDA). In culture, the fungus produced a circular, white to pink colony with pyknotic and linter shaped aerial mycelium. Numerous pycnidia, initially brown and dark at maturity, were embedded in the mycelium, especially in the center of the colony, with a few of them scattered in the edge. The morphological characteristics were consistent with Phoma (2). The ITS1-5.8S-ITS2 region of three isolates were PCR amplified and sequenced with primers ITS1 and ITS4. Sequences (GenBank Accession No. JX885584) showed 99% identity with reference isolates of Peyronellaea glomerata (Corda) Goid (AB470906.1 and HQ380779.1) and Phoma glomerata (Corda) Wollenw. & Hochapfel (EU098115.1). These two species are synonyms (1). To test pathogenicity, nine healthy branches of 3-year-old potted tree peony plants were wound-inoculated with a PDA disk containing pycnidia from an actively growing colony of P. glomerata. Three control branches were inoculated with sterile PDA disks. Each inoculated branch was wrapped in a plastic bag and maintained in a greenhouse at 25 to 28°C. After 3 days, brown patches appeared on inoculated branches and extended by up to 1 cm. Pycnidia identical to those observed in the field and in storage appeared on all inoculated branches 7 days after inoculation. Control branches did not show symptoms. The pathogen was reisolated from inoculated branches, fulfilling Koch's postulates. P. glomerata was reported as the causal agent of withering of flowers and young shoots of grapevines in Yugoslavia (3). To our knowledge, P. glomerata and Botryosphaeria dothidea have always been reported together, causing branch wilting or dieback. To our knowledge, this is the first report of branch blight of tree peony caused by P. glomerata in China. References: (1) M. M. Aveskamp et al. Mycol. Soc. Am. 101:363, 2009. (2) G. H. Boerema et al. Studies in Mycology, 3, 1973. (3) A. Šaric-Sabadoš et al. Atti Ist. bot. Univ. Pavia 18:101, 1960.

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First report of panicle rot caused by Fusarium asiaticum on foxtail millet in China

Plant Disease ◽

10.1094/pdis-04-21-0856-pdn ◽

2021 ◽

Author(s):

Fanxin Kong ◽

Haijin Zhang ◽

Zhi Liu ◽

Guoqiu Chen ◽

Jing Xu

Keyword(s):

Foxtail Millet ◽

Morphological Characteristics ◽

Aerial Mycelium ◽

Its Region ◽

Liaoning Province ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Dark Cycle ◽

First Report ◽

Fusarium Asiaticum

Foxtail millet [ Setaria italica (L.) P. Beauv.] is one of the most important nutritious food crops. It is used for wine and health products in China. In August of 2019, panicle rot symptoms with up to 85% of panicles infected were observed on foxtail millet (cultivar Chaogu 8) in a commercial field located in Chaoyang city of Liaoning Province, China. Typical disease symptoms included brown spots on spikelets at early stages and brown-colored withering and rot of whole panicles at late stages, with the symptoms being more severe at the tip of the panicles. Under high humidity conditions, pink or salmon-colored molds developed on panicles. Symptomatic spikelet pieces were surface-disinfested with 70% ethanol for 1 min followed by 2% NaOCl for 3 min, rinsed with sterilized water for three times, and placed on potato dextrose agar (PDA) medium at 25°C. After 5 days, colonies turned pink to dark red with fluffy aerial mycelium and pigmentation with the age. Ten pure cultures were obtained from single conidia of mycelium grown on carnation leaf agar (CLA) medium at 25°C under a 12-h light-dark cycle using an inoculation needle under stereomicroscope. Macroconidia were hyaline, falcate with foot cells, 3–5 septate and size: 28.5- 44.0 μm × 3.8 - 4.9 μm. Chlamydospores were globose to subglobose (5.4 to 13.8 μm). No microconidia were produced on CLA. Black, ostiolate subglobose perithecia were formed on CLA after one month of incubation at 20°C under a 12-h light-dark cycle. Morphological characteristics of the fungus were in agreement with the description of Fusarium asiaticum (O’Donnell et al. 2004; Leslie and Summerell 2006). To validate this identification, partial translation elongation factor 1 alpha (TEF1-a) gene, and rDNA internal transcribed spacer (ITS) region of five isolates were amplified and sequenced (O’Donnell et al. 2015; White et al.1990). Identical sequences were obtained, and the sequence of one representative isolate (JGF-3) was submitted to GenBank. BLASTn analysis of both TEF sequence (MW685833) and ITS sequence (MW423687), revealed 100% sequence identity with F. asiaticum KT380120 and MT322117, respectively. Pathogenicity test were conducted on cultivar Chaogu 8 of foxtail millet. Inoculum was prepared from the culture of JGF-3 incubated in 2% mung beans juice on a shaker (140 rpm) at 25°C for 48 h. Conidial suspension (5 × 105 conidia per ml) was prepared and sprayed onto the panicles of 20 plants at the initial flowering stage and 20 additional plants that were sprayed with distilled water served as the non-inoculated controls. Treated plants were covered with plastic bags for 48 h and maintained at a greenhouse with day and night temperatures of 26 and 24°C, respectively. Two weeks after inoculation, all inoculated panicles exhibited symptoms similar to the syptoms observed in the field. No symptoms were observed in the non-inoculated control plants. The experiment was repeated twice with similar results. F. asiaticum was reisolated from the inoculated plants and its morphological characteristics matched those of the original isolates; the fungus was not reisolated from the non-inoculated plants. To our knowledge, this is the first report of F. asiaticum causing panicle rot of foxtail millet in China. To date, the disease has been observed to be present in Fuxin and Tieling city of Liaoning Province. Panicle rot can become an important disease in foxtail millet in China. References: O’Donnell, K., et al. 2004. Fungal Genetics and Biology 41: 600. Leslie, J. F., and Summerell, B. A. 2006. The Fusarium laboratory manual. Blackwell Publishing, Ames, pp 176-179. O’ Donnell, K., et al. 2015. Phytoparasitica 43: 583. White, T. J., et al. 1990. Academic Press, San Diego, CA, pp 315-322.

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First Report of Fusarium mangiferae Causing Mango Malformation in China

Plant Disease ◽

10.1094/pdis-12-11-1030 ◽

2012 ◽

Vol 96 (5) ◽

pp. 762-762 ◽

Cited By ~ 8

Author(s):

R.-L. Zhan ◽

S.-J. Yang ◽

F. Liu ◽

Y.-L. Zhao ◽

J.-M. Chang ◽

...

Keyword(s):

Mangifera Indica ◽

Elongation Factor ◽

Morphological Characteristics ◽

Fungal Growth ◽

Aerial Mycelium ◽

Laboratory Manual ◽

First Report ◽

Normal Number ◽

Mango Malformation ◽

Fusarium Mangiferae

Mango (Mangifera indica L.) malformation caused by Fusarium mangiferae has been reported in many mango-growing regions of the world (3). The disease was also observed in Yunnan and Sichuan provinces of China (1). Typical symptoms in seedlings included loss of apical dominance, hyperplasia and hypertrophy of vegetative buds, shortened internodes, and leaves that were more brittle than those of healthy plants. Inflorescences were abnormally branched and thickened, with panicles producing as much as two to five times the normal number of flowers. Flowers in the malformed inflorescence were much more enlarged and crowded than the generally hypertrophied axes of the panicle, thus producing no fruit or aborting early. To identify the pathogen, samples of malformed and healthy mango seedlings were collected from the affected plantings. For isolation, portions of stems were cut into 3- to 4-mm segments, surface disinfested, dried, and then plated on potato dextrose agar and incubated at 25°C. Within 5 days, white, fluffy, aerial mycelium developed. With the aid of an inverted microscope, single conidia were transferred to carnation leaf agar (CLA) medium. After 10 days of incubation, morphological characteristics were found to be identical to those of F. mangiferae (4). Aerial mycelium was white with no pigmentation observed on potato sucrose agar. Pigmentation on rice medium was pink. On CLA medium, conidia grew in branched conidiophores with false heads bearing monophialides or polyphialides. No conidiospores in chains were observed. Microconidia were ovate to long and oval, 0 to 1 septate, and 3.1 to 10.2 × 1.5 to 2.2 μm. Macroconidia are falculate, 3 to 5 septate, and 18 to 38 × 1.8 to 2.4 μm. Chlamydospores were not observed. Pathogenicity studies were conducted with 7-month-old asymptomatic mango seedlings. These seedlings, except for the controls, were inoculated by injection of the isolated fungus in the axillary or apical bud position. A 1-ml spore suspension (1 × 106 spores/ml) was injected slowly into the stems using a microsyringe with three buds per seedling, for a total of 10 seedlings. Typical malformation symptoms developed within 3 to 4 months, and none of the plants inoculated with sterile water resulted in malformation symptoms. Reisolations from the induced malformed shoots yielded the same fungus, and no fungal growth was observed to be growing from the control plants. To confirm identity of the causal fungus, the gene encoding translation elongation factor 1 alpha (EF-1α) was amplified and sequenced (2). The EF-1α sequence was 660 bp long. The sequence (GenBank Accession No. HM068871) was 99.68% similar to sequences of FD_01167 in the Fusarium ID database. On the basis of symptoms, fungal morphology, the EF-1α region sequence, and pathogenicity testing, this fungus was identified as F. mangiferae. To our knowledge, this is the first report of F. mangiferae causing mango malformation in China. This report will establish a foundation for further study of F. mangiferae and effectively addressing the disease. References: (1) X. H. Chen. Pract. Technol. (in Chinese) 6:5, 1992. (2) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (3) J. Kumar et al. Annu. Rev. Phytopathol. 31:217, 1993. (4) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006.

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