First Report of Fusarium oxysporum Causing Coriander Wilt Disease in North China

Coriander or cilantro (Coriandrum sativum L.) is extensively used as a fresh herb in China. During the summer of 2019 in Tianjin, China, coriander plants showed a previously unknown wilt in commercial fields. In severely infected fields, approximately 85% of the plants at vegetative stages were stunted, chlorotic, wilted or dead. Vascular tissues of the main stem and root were discolored. Soil samples were collected from five fields with a history of severe wilt disease in Tianjin since the fall of 2019. Seeds of coriander cultivar “ansemi” were sown in soil from the five fields, and cultured in a greenhouse at 22°C/20°C (12 h /12 h, light/dark) and 75% relative humidity. After 50 days’ cultivation, infected seedlings exibited similar symptoms to those of plants in the field. Twenty symptomic seedlings from each of the collected field soil samples were harvested and washed for 3 min under running water. Then the vascular tissue fragments (3 mm2) of stem and root at the boundary of the symptomatic area were excised, and placed on improved Komada’s medium, which is selective for Fusarium sp. (Komada 1975). After incubation at 25°C for 5 days in the dark, 70% of the isolates generated white to pale pink aerial hyphae on PDA (Fig. S1). Microconidia were single-celled, hyaline, non-septate and ovoid, and measured 5.6 to 14.1 μm long and 2.1 to 3.8 μm wide (n = 40). Macroconidia were three to five-septate, slightly curved at apex, and ranged from 11.2 to 38.6 μm long × 3.2 to 4.5 μm wide (n = 40). Based on morphological characteristics, these fungi were preliminarily identified as F. oxysporum (Leslie and Summerell 2006). For molecular identification, the ITS gene, TEF1-α gene and mtSSU gene of cultures from two representative single spored isolates XC02 and XC03 were amplified and sequenced (White et al. 1990; Carbone and Kohn 1999; Li et al. 1994). The sequences were submitted to GenBank (MT579855 and OK326765 for ITS; MT597425 and OK256882 for TEF1-α; MT587799 and OK330480 for mtSSU). BLASTn analysises indicated that the nucleotide sequences of the three loci of the two isolates were 99.8% to 100% homologous to sequences of F. oxysporum in the NCBI database and Fusarium MLST database. A multilocus phylogenetic tree was drawn via UPGMA analysis of the combined ITS, TEF1-α and mtSSU partial sequence data from Fusarium MLST database (Fig. S2). Pathogenicity of each isolate was tested on ten 15-day-old healthy coriander seedlings in each treatment according to the method of Yang et al. (2020). The first wilt symptoms developed 3 days after inoculation, and 7 to 10 days after inoculation 90% to 100% of the plants were dead. Control plants remained healthy. To fulfill the Koch's postulates, F. oxysporum were reisolated from the diseased tissues and verified based on morphology and sequencing as described above. The experiments were repeated twice with similar results. F. oxysporum has been reported to cause coriander wilt disease in India, Argentina, California (Koike and Gordon 2005), and Italy (Gilardi et al. 2019). To our knowledge, this is the first report of F. oxysporum causing coriander wilt disease in north China. Compared with F. equiseti in the previous report (Yang et al. 2020), F. oxysporum isolates possessed higher separation frequence and stronger pathogenicity to coriander seedlings. Thus attentions should be taken on F. oxysporum causing coriander wilt diseases when developing effective management strategies in north China.

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First Report of Occurrence of Eggplant Wilt Caused by Verticillium dahliae in Korea

Plant Disease ◽

10.1094/pdis.2000.84.10.1152a ◽

2000 ◽

Vol 84 (10) ◽

pp. 1152-1152

Author(s):

S. K. Kim ◽

S. S. Hong ◽

K. W. Kim ◽

E. W. Park

Keyword(s):

Verticillium Dahliae ◽

Solanum Melongena ◽

Morphological Characteristics ◽

Wilt Disease ◽

Potato Dextrose Agar ◽

First Report ◽

Vascular Tissues ◽

Fungal Isolates ◽

Pathogenicity Tests ◽

Root Cutting

A wilt disease occurred on greenhouse-grown eggplants (Solanum melongena L.) at Hanam and Yeojoo, Korea, in 1997. Lower leaves on the 2-month-old wilted eggplants exhibited gradual yellowing, interveinal necrosis, and marginal crinkling and dropped prematurely. Vascular tissues of diseased stems were discolored and turned black. Vertical sections of the stems revealed that the pith had been colonized by the fungus. The disease progressed from lower parts of the plants upward. Incidence of diseased eggplants in greenhouses was 5% on 23 May 1997. Although the incidence increased to 10% on 13 June, it remained constant through early July. Fungal isolates from discolored vascular tissues were initially whitish to cream color on potato-dextrose agar, which turned black due to the formation of microsclerotia. The fungus also produced abundant verticillate conidiophores with phialides and conidia. Based on these cultural and morphological characteristics, the fungus was identified as Verticillium dahliae Klebahn. Pathogenicity tests by root cutting, root dipping, or soil drenching resulted in similar symptoms observed in the naturally infected eggplants. Symptoms were first observed on lower leaves of each eggplant 3 weeks after inoculation. Isolation from symptomatic leaves of the inoculated eggplants yielded V. dahliae. This is the first report of occurrence of Verticillium wilt of eggplant in Korea.

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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 Fusarium oxysporum f. sp. lycopersici Race 3 Causing Fusarium Wilt on Tomato in Korea

Plant Disease ◽

10.1094/pdis-01-13-0073-pdn ◽

2013 ◽

Vol 97 (10) ◽

pp. 1377-1377 ◽

Cited By ~ 4

Author(s):

H.-W. Choi ◽

S. K. Hong ◽

Y. K. Lee ◽

H. S. Shim

Keyword(s):

Fusarium Wilt ◽

Dna Sequences ◽

Vascular Tissue ◽

Elongation Factor ◽

Morphological Characteristics ◽

Spore Suspension ◽

Vascular Bundles ◽

Laboratory Manual ◽

First Report ◽

Blastn Analysis

In July 2010, fusarium wilt symptoms of tomato (Lycopersicon esculentum Mill.) plants were found in two commercial greenhouses in the Damyang area of Korea. Approximately 1% of 7,000 to 8,000 tomato plants were wilted and chlorotic in each greenhouse. The vascular tissue was usually dark brown and the discoloration extended to the apex. Fragments (each 5 × 5 mm) of the symptomatic tissue were surface-sterilized with 1% NaOCl for 1 min, then rinsed twice in sterilized distilled water (SDW). The tissue pieces were placed on water agar and incubated at 25°C for 4 to 6 days. Nine Fusarium isolates were obtained from four diseased plants, of which three isolates were identified as F. oxysporum based on morphological characteristics on carnation leaf agar medium and DNA sequences of the translation elongation factor 1-alpha (EF-1α) gene (2). Macroconidia were mostly 3- to 5-septate, slightly curved, and 28 to 53 × 2.8 to 5.2 μm. Microconidia were abundant, borne in false heads or short monophialides, generally single-celled, oval to kidney shaped, and 5 to 23 × 3 to 5 μm. Chlamydospores were single or in short chains. The EF-1α gene was amplified from three isolates by PCR assay using ef1 and ef2 primers (3), and the amplification products were sequenced. The nucleotide sequences obtained were deposited in GenBank (Accession Nos. KC491844, KC491845, and KC491846). BLASTn analysis showed 99% homology with the EF-1α sequence of F. oxysporum f. sp. lycopersici MN-24 (HM057331). Pathogenicity tests and race determination were conducted using root-dip inoculation (4) on seedlings of tomato differential cultivars: Ponderosa (susceptible to all races), Momotaro (resistant to race 1), Walter (resistant to races 1 and 2), and I3R-1 (resistant to all races). A spore suspension was prepared by flooding 5-day-old cultures on potato dextrose agar with SDW. Plants at the first true-leaf stage were inoculated by dipping the roots in the spore suspension (1 × 106 conidia/ml) for 10 min. Inoculated plants were transplanted into pots containing sterilized soil, and maintained in the greenhouse at 25/20°C (12/12 h). Twenty-four seedlings of each cultivar were arranged into three replications. An equal number of plants of each cultivar dipped in water were used as control treatments. Disease reaction was evaluated 3 weeks after inoculation, using a disease index on a scale of 0 to 4 (0 = no symptoms, 1 = slightly swollen and/or bent hypocotyl, 2 = one or two brown vascular bundles in the hypocotyl, 3 = at least two brown vascular bundles and growth distortion, 4 = all vascular bundles brown and the plant either dead or very small and wilted). All isolates caused symptoms of fusarium wilt on all cultivars except I3R-1, indicating that the isolates were race 3. The pathogen was reisolated from the discolored vascular tissue of symptomatic plants. Control plants remained asymptomatic, and the pathogen was not reisolated from the vascular tissue. Fusarium wilt of tomato caused by isolates of F. oxysporum f. sp. lycopersici races 1 and 2 has been reported previously; however, race 3 has not been reported in Korea (1). To our knowledge, this is the first report of isolates of F. oxysporum f. sp. lycopersici race 3 on tomato in Korea. References: (1) O. S. Hur et al. Res. Plant Dis. 18:304, 2012 (in Korean). (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (3) K. O'Donnell et al. Proc. Nat. Acad. Sci. 95:2044, 1998. (4) M. Rep et al. Mol. Microbiol. 53:1373, 2004.

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First Report of Dolabra nepheliae Associated with Corky Bark Disease of Rambutan and Pulasan in Honduras

Plant Disease ◽

10.1094/pdis-01-12-0081-pdn ◽

2012 ◽

Vol 96 (5) ◽

pp. 765-765 ◽

Cited By ~ 2

Author(s):

A. Rossman ◽

J. Melgar ◽

D. Walker ◽

A. Gonzales ◽

T. Ramirez ◽

...

Keyword(s):

Puerto Rico ◽

Sequence Data ◽

Management Strategies ◽

Pairwise Comparison ◽

Its Region ◽

Its Sequence ◽

Centraalbureau Voor ◽

First Report ◽

Intergenic Spacers ◽

Old Trees

In the last decade, rambutan (Nephelium lappaceum L., Sapindaceae) and pulasan (N. mutabile Blume) have been cultivated in Honduras to produce exotic fruits for export to North America (2). Recently, a disease was observed that produces dark brown to black fissured cankers from 1 to 3 cm long and 1 to 4 cm wide. The infected bark tissue becomes swollen with the middle region 3 to 8 mm thick. Symptoms appear when the trees are approximately 3 years old. As the trees mature, the cankers increase in size and weaken the branches, often resulting in breakage with the weight of the fruit causing substantial plant damage and fruit loss. In August 2010, fissured branch samples of rambutan and pulasan were collected from 6- to 8-year-old trees from the Humid Tropical Demonstrative Agroforestry Center in Honduras, Atlantida, La Masica (15°33′47.4″N, 87°05′2.5″W, elevation 106 m). A fungus associated with the cankers was identified as Dolabra nepheliae. It produces black, stipitate, elongate ascomata, 312 to 482 × 250 to 281 μm with broadly cylindric, bitunicate asci, 120 to 138 × 11.2 to 15.0 μm, and filiform, hyaline ascospores, 128 to 135 × 2.8 to 3.2 μm. Fungi from rambutan and pulasan were isolated on cornmeal agar plus 0.5% dextrose and antibiotics. On potato dextrose agar, the ascospores produced slow-growing colonies, 5 mm per week. In culture, isolates from both hosts produced pycnidia with elongated, slightly to strongly curved or S-shaped, hyaline conidia, 22.8 to 46.4 × 2.8 to 3.7 μm. This fungus was first reported on rambutan and pulasan from Malaysia (1,4), and later reported on rambutan and litchi in Hawaii and Puerto Rico (3). To our knowledge, this is the first report of D. nepheliae on pulasan and rambutan from Honduras. Specimens have been deposited at the U.S. National Fungus Collections (BPI 882442 on N. lappaceum and BPI 882443 on N. mutabile). Cultures were deposited at the Centraalbureau voor Schimmelcultures (CBS) as CBS 131490 on N. lappaceum and CBS 131491 on N. mutabile. Sequences of the internal transcribed spacer (ITS) region including ITS1, 5.8S, and ITS2 intergenic spacers were deposited in GenBank (Accession No. JQ004281 on N. lappaceum and Accession No. JQ004280 on N. mutabile). A BLAST search and pairwise comparison using the GenBank web server were used to compare ITS sequence data and recovered the following results: (i) CBS 131490 on N. lappaceum is 99% (538 of 544) identical to D. nepheliae CBS 123297 on Litchi chinensis from Puerto Rico; and (ii) CBS 131491 on N. mutabile is 99% (527 of 533) identical to the same strain of D. nepheliae. On the basis of the ITS sequence data, the isolates from Honduras were confirmed as the same species, D. nepheliae from Puerto Rico. Efforts to develop resistant germplasm and management strategies to control this disease have been initiated. References: (1) C. Booth and W. P. Ting. Trans. Brit. Mycol. Soc. 47:235, 1964. (2) T. Ramírez et al. Manual Para el Cultivo de Rambutan en Honduras. Fundación Hondureña de Investigación Agrícola. La Lima, Cortes, Honduras, 2003. (3) A. Y. Rossman et al. Plant Dis. 91:1685, 2007. (4) H. Zalasky et al. Can. J. Bot. 49:559, 1971.

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First Report of Fusarium Wilt of Cilantro Caused by Fusarium oxysporum in California

Plant Disease ◽

10.1094/pd-89-1130a ◽

2005 ◽

Vol 89 (10) ◽

pp. 1130-1130 ◽

Cited By ~ 3

Author(s):

S. T. Koike ◽

T. R. Gordon

Keyword(s):

Fusarium Oxysporum ◽

Fusarium Wilt ◽

Vascular Tissue ◽

Fusarium Species ◽

Wilt Disease ◽

Apium Graveolens ◽

Peat Moss ◽

State University ◽

First Report ◽

Vascular Discoloration

Cilantro, or coriander (Coriandrum sativum), is a leafy vegetable in the Apiaceae and is grown commercially in California primarily for use as a fresh herb. During 2002 and 2003 in coastal California (Santa Barbara County), commercial cilantro fields showed symptoms of a wilt disease. Affected plants grew poorly and were stunted. Lower foliage turned yellow with reddish tinges, and plants wilted during warmer times of the day. The main stem, crown, and taproot exhibited vascular discoloration that was reddish to light brown. As disease progressed, plants eventually died. For both years, the disease distribution was limited to isolated small patches (each patch measuring less than 1 m2 in area). A fungus was consistently isolated from symptomatic vascular tissue in crowns and taproots. On the basis of colony and conidial morphology, the isolates were identified as Fusarium oxysporum (2). No other fungi or bacteria were recovered from these plants. To test pathogenicity, suspensions containing 1 × 106 conidia/ml were prepared for five isolates. The roots of 30-day-old cilantro plants of four cultivars (30 plants each of Festival, Leisure, Santo, and LSO 14) were clipped and then soaked in the suspensions for 20 min. The roots of 30 plants of each cultivar were soaked in water as a control. Plants were repotted into new redwood bark + peat moss rooting medium and maintained in a greenhouse setting at 24 to 26°C. After 1 month, 95% or more of the inoculated plants showed yellowing and vascular discoloration symptoms similar to those seen in the field. F. oxysporum was reisolated from all inoculated plants. The four cilantro cultivars did not show differences in disease severity. Control plants showed no symptoms, and the fungus was not recovered from these plants. The experiment was repeated and the results were the same. Experiments also were conducted to determine if cilantro isolates could cause disease in celery (Apium graveolens var. dulce). Celery transplants and cilantro seedlings were prepared and inoculated as described above. However, after 2 months, celery plants did not show any disease symptoms, while the cilantro developed wilt symptoms and eventually died. A Fusarium wilt disease has been reported on coriander in Argentina and India where the pathogen was named F. oxysporum f. sp. coriandrii (1,3). To our knowledge, this is the first report of Fusarium wilt of cilantro in California. References: (1) M. Madia et al. Fitopatologia 34:155, 1999. (2) P. E. Nelson et al. Fusarium species: An Illustrated Manual for Identification. Pennsylvania State University Press, University Park, 1983. (3) U. S. Srivastava. Indian Phytopathol. 22:406, 1969.

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First Report of Powdery Mildew Caused by Blumeria graminis f. sp. bromi on Bromus catharticus in China

Plant Disease ◽

10.1094/pdis-09-20-1983-pdn ◽

2020 ◽

Author(s):

Mo Zhu ◽

Jie Ji ◽

Xiao Duan ◽

Wenqi Shi ◽

YongFang Li

Keyword(s):

Powdery Mildew ◽

Sequence Data ◽

Morphological Characteristics ◽

Signs And Symptoms ◽

The United States ◽

Blumeria Graminis ◽

Henan Province ◽

Width Ratio ◽

Causal Organism ◽

First Report

Bromus catharticus, rescuegrass, is a brome grass that has been cultivated for herbage production, and been widely naturalized in many provinces of China, including Henan province. During April and May 2020, powdery mildew was found on leaves of Br. catharticus on the campus of Henan Normal University, Xinxiang city (35.3°N; 113.9°E), Henan Province, China. Abundant white or grayish irregular or coalesced circular powdery colonies were scattered on the adaxial surface of leaves and 70% of the leaf areas were affected. Some of the infected leaves either were chlorotic or senescent. About 60% of the observed plants showed powdery mildew symptoms. Conidiophores (n = 25) were 32 to 45 μm × 7 to 15 μm and composed of foot cells and conidia (mostly 6 conidia) in chains. Conidia (n = 50) were 25 to 35 μm × 10 to 15 μm, on average 30 × 13 μm, with a length/width ratio of 2.3. Chasmothecia were not found. Based on these morphologic characteristics, the pathogen was initially identified as Blumeria graminis f. sp. bromi (Braun and Cook 2012; Troch et al. 2014). B. graminis mycelia and conidia were collected, and total genomic DNA was extracted (Zhu et al. 2019). The rDNA internal transcribed spacer (ITS) region was amplified with primer pairs ITS1/ITS4. The amplicon was cloned and sequenced. The sequence (574 bp) was deposited into GenBank under Accession No. MT892940. BLASTn analysis revealed that MT892940 was 100% identical to B. graminis f. sp. bromi on Br. catharticus (AB000935, 550 of 550 nucleotides) (Takamatsu et al. 1998). Phylogenetic analysis of MT892940 and ITS of other B. graminis ff. spp. clearly indicated least two phylogenetically distinct clades of B. graminis f. sp. bromi and that MT892940 clustered with the Takamatsu vouchers. Leaf surfaces of five healthy plants were fixed at the base of a settling tower and then inoculated by blowing conidia from diseased leaves using pressurized air. Five non-inoculated plants served as controls. The inoculated and non-inoculated plants were maintained separately in two growth chambers (humidity, 60%; light/dark, 16 h/8 h; temperature, 18℃). Thirteen- to fifteen-days after inoculation, B. graminis signs and symptoms were visible on inoculated leaves, whereas control plants remained asymptomatic. The pathogenicity assays were repeated twice with the same results. The observed signs and symptoms were morphologically identical to those of the originally infected leaves. Accordingly, the causal organism of the powdery mildew was confirmed as B. graminis f. sp. bromi by morphological characteristics and ITS sequence data. B. graminis has been reported on Br. catharticus in the United States (Klingeman et al. 2018), Japan (Inuma et al. 2007) and Argentina (Delhey et al. 2003). To our best knowledge, this is the first report of B. graminis on Br. catharticus in China. Since hybridization of B. graminis ff. spp. is a mechanism of adaptation to new hosts, Br. catharticus may serve as a primary inoculum reservoir of B. graminis to infect other species (Menardo et al. 2016). This report provides fundamental information for the powdery mildew that can be used to develop control management of the disease in Br. catharticus herbage production.

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First report of preharvest fruit rot of ‘Pink Lady’ apples caused by Colletotrichum fructicola in Italy

Plant Disease ◽

10.1094/pdis-11-20-2404-pdn ◽

2021 ◽

Author(s):

Marcel Wenneker ◽

Khanh Pham ◽

Engelien Kerkhof ◽

Dalphy O.C. Harteveld

Keyword(s):

Species Complex ◽

Management Strategies ◽

Morphological Characteristics ◽

Late Summer ◽

Fruit Rot ◽

Colletotrichum Acutatum ◽

Apple Orchards ◽

First Report ◽

Bitter Rot ◽

Colletotrichum Fructicola

In late summer 2019, a severe outbreak of fruit rot was observed in commercial ‘Pink Lady’ apple orchards (>20 ha in total) in the region Emilia-Romagna (Northern Italy). The symptoms on the fruit appeared as small circular red to brown lesions. Disease incidences of over 50% of the fruits were observed. To isolate the causal agent, 15 affected apples were collected and small portions of fruit flesh were excised from the lesion margin and placed on potato dextrose agar (PDA). The plates were incubated at 20°C in the dark, and pure cultures were obtained by transferring hyphal tips on PDA. The cultures showed light to dark gray, cottony mycelium, with the underside of the culture being brownish and becoming black with age. Conidia (n=20) were cylindrical, aseptate, hyaline, rounded at both ends, and 12.5 to 20.0 × 5.0 to 7.5 μm. The morphological characteristics were consistent with descriptions of Colletotrichum species of the C. gloeosporioides species complex, including C. fructicola (Weir et al. 2012). The identity of two representative isolates (PinkL2 & PinkL3) from different apples was confirmed by means of multi-locus gene sequencing. Genomic DNA was extracted using the LGC Mag Plant Kit (Berlin, Germany) in combination with the Kingfisher method (Waltham, USA). Molecular identification was conducted by sequencing the ITS1/ITS4 region and partial sequences of four other gene regions: chitin synthase (CHS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), and beta-tubulin (TUB). The sequences have been deposited in GenBank under accession numbers MT421924 & MT424894 (ITS), MT424612 & MT424613 (CHS), MT424616 & MT424617 (GAPDH), MT424614 & MT424615 (ACT), and MT424620 & MT424621 (TUB). MegaBLAST analysis revealed that our ITS sequences matched with 100% identity to Colletotrichum fructicola (Genbank JX010177). The CHS, GAPDH, ACT and TUB sequences of both isolates were 100% identical with C. fructicola culture collection sequences in Genbank (JX009807, JX009923, JX009436 and JX010400, respectively), confirming the identity of these isolates as C. fructicola. Koch's postulates were performed with 10 mature ‘Pink Lady’ apples. Surface sterilized fruit were inoculated with 20 μl of a suspension of 105 conidia ml–1 after wounding with a needle. The fruits were incubated at 20˚C at high relative humidity. Typical symptoms appeared within 4 days on all fruit. Mock-inoculated controls with sterile water remained symptomless. The fungus was reisolated and confirmed as C. fructicola by morphology and sequencing of all previously used genes. Until recently the reported causal agents of bitter rot of apple in Europe belong to the Colletotrichum acutatum species complex (Grammen et al. 2019). C. fructicola, belonging to C. gloeosporioides species complex, is known to cause bitter rot of apple in the USA, Korea, Brazil, and Uruguay (Kim et al. 2018; Velho et al. 2015). There is only one report of bitter rot associated with C. fructicola on apple in Europe (France) (Nodet et al. 2019). However, C. fructicola is also the potential agent of Glomerella leaf spot (GLS) of apple (Velho et al. 2015; 2019). To the best of our knowledge this is the first report of C. fructicola on apples in Italy. It is important to stress that the C. gloeosporioides species complex is still being resolved and new species on apple continue to be identified, e.g. C. chrysophilum that is very closely related to C. fructicola (Khodadadi et al. 2020). Given the risks of this pathogen the presence of C. fructicola in European apple orchards should be assessed and management strategies developed.

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First Report of Fusarium ipomoeae Causing Peanut leaf Spot in China

Plant Disease ◽

10.1094/pdis-01-21-0226-pdn ◽

2021 ◽

Author(s):

Manlin Xu ◽

Xia Zhang ◽

Jing Yu ◽

zhiqing Guo ◽

Ying Li ◽

...

Keyword(s):

Leaf Spot ◽

Genomic Dna ◽

Block Design ◽

Management Strategies ◽

Morphological Characteristics ◽

Ethanol Solution ◽

Distilled Water ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

First Report

Peanut (Arachis hypogaea L.) is one of the most economically important crops as an important source of edible oil and protein. In August 2020, circular to oval-shaped brown leaf spots (2-6 mm in diameter) with well-defined borders surrounded by a yellow margin were observed on peanut plant leaves in Laixi City, Shandong Province, China. Symptomatic plants randomly distributed in the field, the incidence was approximately 5%. Leave samples were collected consisted of diseased tissue and the adjacent healthy tissue. The samples were dipped in a 70% (v/v) ethanol solution for 30 s and then soaked in a 0.1% (w/v) mercuric chloride solution for 60 s. The surface-sterilized tissues were then rinsed three times with sterile distilled water, dried and placed on Czapek Dox agar supplemented with 100 μg/ml of chloramphenicol. The cultures were incubated in darkness at 25 °C for 3–5 days. Fungal colonies were initially white and radial, turning to orange-brown in color, with abundant aerial mycelia. Macroconidia were abundant, 4 to 7 septate, with a dorsiventral curvature, and were 3.3–4.5 × 18.5–38.1 μm (n=100) in size; microconidia were absent; chlamydospores were produced in chains or clumps, ellipsoidal to subglobose, and thick walled. The morphological characteristics of the conidia were consistent with those of Fusarium spp. To identify the fungus, an EasyPure Genomic DNA Kit (TransGEN, Beijing, China) was used to extract the total genomic DNA from mycelia. The internal transcribed spacer region (ITS rDNA) and the translation elongation factor 1-α gene (TEF1) were amplified with primers ITS1/ITS4 (White et al. 1990) and EF1/EF2 (O’Donnell et al. 1998), respectively. Based on BLAST analysis, sequences of ITS (MT928727) and TEF1 (MT952337) showed 99.64% and 100% similarity to the ITS (MT939248.1), TEF1 (GQ505636.1) of F. ipomoeae isolates. Sequence analysis confirmed that the fungus isolated from the infected peanut was F. ipomoeae (Xia et al. 2019). The pathogenicity of the fungus was tested in the greenhouse. Twenty two-week-old peanut seedlings (cv. Huayu20) grown in 20-cm pots (containing autoclaved soil) were sprayed with a conidial suspension (105 ml−1) from a 15-day-old culture. Control plants were sprayed with distilled water. The experiment was conducted as a randomized complete block design, and placed at 25 °C under a 12-h photoperiod with 90% humidity. Symptoms similar to those in the field were observed on leaves treated with the conidial suspension ten days after inoculation, but not on control plants. F. ipomoeae was re-isolated from symptomatic leaves but not from the control plants. Reisolation of F. ipomoeae from inoculated plants fulfilled Koch's postulates. To our knowledge, this is the first report of F. ipomoeae causing peanut leaf spot in China. Our report indicates the potential spread of this pathogen in China and a systematic survey is required to develop effective disease management strategies.

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First report of Fusarium cf. longipes associated with maize stalk rot in China

Plant Disease ◽

10.1094/pdis-06-21-1149-pdn ◽

2021 ◽

Author(s):

Shengbo Han ◽

Yanyong Cao ◽

Jie Zhang ◽

Jie Wang ◽

Lili Zhang ◽

...

Keyword(s):

Sequence Data ◽

Fusarium Species ◽

Apical Cell ◽

Morphological Characteristics ◽

Liaoning Province ◽

Bootstrap Support ◽

Flowering Stage ◽

Single Spore ◽

First Report ◽

Stalk Rot

In a field survey from 2017 to 2019, Fusarium stalk rot symptoms including discolored, disintegrated stalk pith tissues and lodged plants were observed in maize hybrid lines Fuyu1611, Jidan66, and Danyu8439 grown in fields in Anshan (40o49′39′′N, 122 o34′6′′E), Liaoning province. Its incidence ranged from 15% to 20% and caused a yield loss of up to 30%. Infected pieces of stem tissues were dissected and then sterilized with 1% NaOCl for 1 min, 70% ethanol for 1 min, rinsed 3 times with sterilized ddH2O, and dried with filter paper in hood. Three pieces were placed onto Potato dextrose agar (PDA) and incubated at 25 °C for 5 days. The colonies were single-spore subcultured on PDA at 25 °C for 2 weeks (Leslie and Summerell 2006). Morphological features were observed on PDA and carnation leaf agar (CLA). The average mycelial growth rate was 4.5 to 10.3 mm/day at 25 °C on PDA. The colonies produced aerial mycelia, varying from dense white to grayish-rose, and secreted red pigments in the agar (Fig. 1A; 1B). Macroconidia produced on CLA were long and relatively slender, commonly 4- to 7-septate, averaging 85.6 × 5.2 μm, with thick walls and pronounced dorsiventral curvature with a distinctly foot-shaped and elongated basal cell and an apical cell that was whip-like (Fig. 1C). Microconidia were rarely observed on PDA or CLA. Morphological characteristics of the isolates were similar to the features of Fusarium longipes as previously described (Leslie and Summerell 2006). The portions of three phylogenic loci (EF1-α, RPB1, RPB2) were PCR amplified using the primer pairs EF1/EF2 (O'Donnell et al, 1998), lonR1F/lonR1R (5-TTTTCCTCACCAAGGAGCAGATCATG-3 and 5-CCAATGGACTGGGCAGCCAAAACGCC-3) and lonR2F/lonR2R (5-TATACATTTGCCTCCACTCTTTCCCAT-3 and 5-CGGAGCTTGCGTCCGGTGTGGCCGTTG-3) and sequenced. The consensus sequences were submitted to GenBank (MT513215 and MT997083 for TEF, MT513213 and MT997088 for RPB1; MT513214 and MW020572 for RPB2). BLASTn searches indicated that the nucleotide sequences of the three loci of the two isolates shared 94.52% to 99.69% identity with sequences of F. longipes strains deposited in the GenBank, Fusarium-ID and Fusarium MLST databases (Supplementary Table 1, 3, 4). A phylogram inferred via maximum likelihood analysis of the combined EF-1α, RPB1, RPB2 partial sequence data of Fusarium species (Supplementary Table 2) was inferred using the CIPRIES website (https://www.phylo.org). Isolates LNAS-05-A and LNAS-09-A clustered with F. longipes, with 98% bootstrap support (Fig. 2). Pathogenicity tests were conducted on three-leaf-stage seedlings and flowering-stage c.v. Zhengdan958 and B104 plants according to previously described methods (Ye et al., 2013; Zhang et al. 2016) with minor modifications. Three days after the roots of the seedlings were inoculated with 1 × 106 macroconidia solution, the leaves and stems exhibited typical wilt symptoms (Fig. 1D). Twenty flowering-stage maize plants were drilled individually at the second or third node above the soil using an electric drill (Bosch TSR1080-2-Li) to create a hole (8 mm in diameter). An approximately 0.5 mL mycelia plug (125 mL homogenized hyphal mats + 75 mL sterilized ddH2O) was injected into the hole and covered with Vaseline. Sterilized PDA plugs were used as a control. The stalk tissue of the split internodes turned dark brown and the brown area expanded above and below the injection site by 14 dpi. All of the inoculated plants developed characteristic stalk rot symptoms, whereas no symptoms were observed in the controls (Fig. 1E). The pathogen was re-isolated, and its identity was confirmed by sequencing the above mentioned loci. F. longipes was generally regarded as a tropical Fusarium species (Leslie and Summerell 2006). This is the first report that F. cf. longipes can cause stalk rot of maize under filed condition in a temperate, typical corn belt region of China.

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First Report of Anthracnose Disease on Young Stems of Bawanghua (Hylocereus undatus) Caused by Colletotrichum gloeosporioides in China

Plant Disease ◽

10.1094/pdis-06-13-0609-pdn ◽

2014 ◽

Vol 98 (7) ◽

pp. 991-991 ◽

Cited By ~ 2

Author(s):

W. J. Ma ◽

X. Yang ◽

X. R. Wang ◽

Y. S. Zeng ◽

M. D. Liao ◽

...

Keyword(s):

Colletotrichum Gloeosporioides ◽

Sequence Data ◽

Southern China ◽

Morphological Characteristics ◽

Its Region ◽

Pathogenic Fungi ◽

First Report ◽

Guangzhou City ◽

Anthracnose Disease ◽

Hylocereus Undatus

Hylocereus undatus widely grows in southern China. Some varieties are planted for their fruits, known as dragon fruits or Pitaya, while some varieties for their flowers known as Bawanghua. Fresh or dried flowers of Bawanghua are used as routine Chinese medicinal food. Since 2008, a serious anthracnose disease has led to great losses on Bawanghua flower production farms in the Baiyun district of Guangzhou city in China. Anthracnose symptoms on young stems of Bawanghua are reddish-brown, sunken lesions with pink masses of spores in the center. The lesions expand rapidly in the field or in storage, and may coalesce in the warm and wet environment in spring and summer in Guangzhou. Fewer flowers develop on infected stems than on healthy ones. The fungus overwinters in infected debris in the soil. The disease caused a loss of up to 50% on Bawanghua. Putative pathogenic fungi with whitish-orange colonies were isolated from a small piece of tissue (3 × 3 mm) cut from a lesion margin and cultured on potato dextrose agar in a growth chamber at 25°C, 80% RH. Dark colonies with acervuli bearing pinkish conidial masses formed 14 days later. Single celled conidia were 11 to 18 × 4 to 6 μm. Based on these morphological characteristics, the fungi were identified as Colletotrichum gloeosporioides (Penz.) Penz. & Sacc (2). To confirm this, DNA was extracted from isolate BWH1 and multilocus analyses were completed with DNA sequence data generated from partial ITS region of nrDNA, actin (ACT) and glutamine synthetase (GS) nucleotide sequences by PCR, with C. gloeosporioides specific primers as ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) / CgInt (5′-GGCCTCCCGCCTCCGGGCGG-3′), GS-F (5′-ATGGCCGAGTACATCTGG-3′) / GS-R (5′-GAACCGTCGAAGTTCCAC-3′) and actin-R (5′-ATGTGCAAGGCCGGTTTCGC-3′) / actin-F (5′-TACGAGTCCTTCTGGCCCAT-3′). The sequence alignment results indicated that the obtained partial ITS sequence of 468 bp (GenBank Accession No. KF051997), actin sequence of 282 bp (KF712382), and GS sequence of 1,021 bp (KF719176) are 99%, 96%, and 95% identical to JQ676185.1 for partial ITS, FJ907430 for ACT, and FJ972589 for GS of C. gloeosporioides previously deposited, respectively. For testing its pathogenicity, 20 μl of conidia suspension (1 × 106 conidia/ml) using sterile distilled water (SDW) was inoculated into artificial wounds on six healthy young stems of Bawanghua using sterile fine-syringe needle. Meanwhile, 20 μl of SDW was inoculated on six healthy stems as a control. The inoculated stems were kept at 25°C, about 90% relative humidity. Three independent experiments were carried out. Reddish-brown lesions formed after 10 days, on 100% stems (18 in total) inoculated by C. gloeosporioides, while no lesion formed on any control. The pathogen was successfully re-isolated from the inoculated stem lesions on Bawanghua. Thus, Koch's postulates were fulfilled. Colletotrichum anthracnose has been reported on Pitaya in Japan (3), Malaysia (1) and in Brazil (4). To our knowledge, this is the first report of anthracnose disease caused by C. gloeosporioides on young stems of Bawanghua (H. undatus) in China. References: (1) M. Masyahit et al. Am. J. Appl. Sci. 6:902, 2009. (2) B. C. Sutton. Page 402 in: Colletotrichum Biology, Pathology and Control. J. A. Bailey and M. J. Jeger, eds. CAB International, Wallingford, UK, 1992. (3) S. Taba et al. Jpn. J. Phytopathol. 72:25, 2006. (4) L. M. Takahashi et al. Australas. Plant Dis. Notes 3:96, 2008.

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