First Report of Leaf Spot of Lettuce (Lactuca sativa L.) Caused by Phoma tropica in Italy

Lettuce (Lactuca sativa L.) is widely grown in Italy, with the production for the preparation of ready-to-eat salads becoming increasingly important. During the spring of 2011, a previously unknown leaf spot was observed on L. sativa plants, cv Rubia, grown in several plastic tunnels in Lumbardy (northern Italy), 20 to 25 days after sowing. Thirty to forty per cent of leaves of the plants growing in the part of the tunnel with the highest relative humidity were affected. Leaves of infected plants showed extensive, irregular, dark brown, necrotic lesions with a chlorotic halo. Lesions initially ranged from 0.5 to 3 mm, then eventually coalesced, reaching 2 to 3 cm, showing a well-defined, dark brown border. Affected leaves senesced and withered. The crown was not affected by the disease. Diseased tissue was excised, immersed in a solution containing 1% sodium hypochlorite for 60 s, rinsed in water, then cultured on potato dextrose agar (PDA), amended with 25 mg/l of streptomycin sulphate. After 5 days, a fungus developed, producing a greenish grey mycelium with a white border when incubated under 12 h/day of fluorescent light at 21 to 23°C. In order to favor the production of conidia, the fungus was transferred on malt extract agar (MA) and maintained under 12 h/day of fluorescent light at 22°C. After 15 days, black pycnidia, 175 to 225 μm, developed, with hyaline, elliptical, unicellular conidia, measuring 3.21 to 6.7 × 1.08 to 3.2 (average 5.5 × 1.9) μm. On the basis of these morphological characteristics, the fungal causal agent of the disease could be related to the genus Phoma (2). The internal transcribed spacer (ITS) region of rDNA of the isolate PHT30 was amplified using the primers ITS1/ITS4 and sequenced. BLAST analysis (1) of the 466-bp segment showed a 99% similarity with the sequence of Phoma tropica (GenBank Accession No. JF923820.1). The nucleotide sequence has been assigned the GenBank Accession No. JQ954396. Pathogenicity tests were performed by spraying healthy 20-day-old lettuce plants, cv Rubia, with a spore suspension (1 × 105 conidia/ml) prepared from 14-day-old colonies of the strain PHT30 grown on MA cultures. Plants inoculated with water alone served as controls. Ten plants per isolate were used. Plants were covered with plastic bags for 5 days after inoculation and maintained in a growth chamber at 20°C and 80% relative humidity. The first foliar lesions, similar to those occurring on the naturally infected plants, developed on leaves 12 days after inoculation. Control plants remained healthy. The pathogen was consistently reisolated from leaf lesions. The pathogenicity test was completed twice. To our knowledge, this is the first report of the presence of P. tropica on lettuce in Italy as well as worldwide. In the United States, the presence of P. exigua was reported in 2006 (3). The economic importance of the disease at present is limited, probably also because symptoms can be confused with those caused by Botrytis cinerea. However, P. tropica could become a more significant problem because of the importance of the crop. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) G. H. Boerema. Trans. Br. Mycol. Soc. 67:289, 1976. (3) S. Y. Koike. Plant Dis. 90:1268, 2006.

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First Report of Leaf Spot of Milky Bellflower (Campanula lactiflora) Caused by a Phoma sp. in Italy

Plant Disease ◽

10.1094/pdis-94-5-0638a ◽

2010 ◽

Vol 94 (5) ◽

pp. 638-638

Author(s):

A. Garibaldi ◽

D. Bertetti ◽

C. Pellegrino ◽

M. L. Gullino

Keyword(s):

United States ◽

Leaf Spot ◽

Morphological Characteristics ◽

Its Region ◽

The United States ◽

Fluorescent Light ◽

Herbaceous Plant ◽

Pathogenicity Test ◽

First Report ◽

Perennial Herbaceous Plant

Campanula lactiflora (milky bellflower), a perennial herbaceous plant in the Campanulaceae, is used in park and gardens and sometimes cultivated for cut flower production. In June 2008, a previously unknown leaf spot was observed on C. lactiflora ‘New Hybrids’ plants from an experimental nursery located near Carmagnola (Torino, northern Italy). Leaves of infected plants showed extensive and irregular, dark brown, necrotic lesions that were slightly sunken with well-defined borders. Lesions initially ranged from 0.5 to 3 mm, eventually coalesced, and covered the entire leaf. Black pycnidia (107 to 116 μm in diameter) containing hyaline, ellipsoid, nonseptate conidia measuring 3.7 to 4.7 × 1.2 to 2.0 (average 4.3 × 1.6) μm were observed. On the basis of these morphological characteristics, the fungal causal agent of the disease could be related to the genus Phoma. In some cases, the basal leaves turned completely necrotic and the plant died. The disease affected 50% of plants. Diseased tissue was excised, immersed in a solution containing 1% sodium hypochlorite for 2 to 3 s, rinsed in water, and then cultured on potato dextrose agar (PDA) medium. A fungus developed that produced a greenish gray mycelium with a white border when incubated under 12 h/day of fluorescent light at 22 to 25°C. The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS4/ITS6 and sequenced. BLAST analysis (1) of the 459-bp segment showed a 100% similarity with the sequence of a Didymella sp. (synonym Mycosphaerella), anamorphic stage of Phoma spp. The nucleotide sequence has been assigned GenBank Accession No. GU128503. Pathogenicity tests were performed by placing 8-mm-diameter mycelial disks removed from PDA cultures of the fungus isolated from infected plants on leaves of healthy potted 4-month-old C. lactiflora ‘New Hybrids’ plants. Eight disks were placed on each plant. Plants inoculated with PDA alone served as controls. Six plants per treatment were used. Plants were covered with plastic bags for 4 days after inoculation and maintained in a growth chamber with daily average temperatures ranging between 23 and 24°C. The first foliar lesions developed on leaves 5 days after inoculation, and after 8 days, 80% of leaves were severely infected. Control plants remained healthy. A Didymella sp. was consistently reisolated from leaf lesions. The pathogenicity test was completed twice. To our knowledge, this is the first report of the presence of a Didymella sp. on C. lactiflora in Italy. Mycosphaerella campanulae and M. minor were reported on C. americana and C. lasiocarpa in the United States (2). The economic importance of the disease currently is limited, but could become a more significant problem in the future if the cultivation of this species becomes more widespread. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN, 1989.

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First Report of Leaf Spot of Orange Coneflower (Rudbeckia fulgida) Caused by a Phoma sp. in Italy

Plant Disease ◽

10.1094/pdis-94-6-0788a ◽

2010 ◽

Vol 94 (6) ◽

pp. 788-788

Author(s):

A. Garibaldi ◽

D. Bertetti ◽

M. T. Amatulli ◽

M. L. Gullino

Keyword(s):

Leaf Spot ◽

Morphological Characteristics ◽

Its Region ◽

The United States ◽

Pathogenicity Test ◽

First Report ◽

Humidity Chamber ◽

Pathogenicity Tests ◽

Symptomatic Tissue ◽

White Mycelium

Rudbeckia fulgida (orange coneflower) is an herbaceous species (Asteraceae) grown in full sun in flower beds and borders in gardens. In the summer of 2009, a previously unknown leaf spot was observed on R. fulgida plants in three private gardens located near Biella (northern Italy). Leaves of infected plants showed extensive and irregular, dark brown, necrotic lesions that were slightly sunken with a well-defined border. Lesions initially ranged from 0.5 to 3 mm in diameter and eventually coalesced to cover the entire leaf, which curled without falling. At a later stage, stems were also affected, causing death of the plant. The disease affected 90% of plants. Dark brown pycnidia, 68 to 195 × 60 to 165 (average 135 × 117) μm in diameter, containing hyaline (light gray in mass), and ellipsoid, nonseptate conidia measuring 4.0 to 7.0 × 2.4 to 3.5 (average 5.4 × 3.0) μm were observed on symptomatic tissue. On the basis of these morphological characteristics, the fungus was related to the genus Phoma. Diseased tissue was excised from the margin of lesions, immersed in a solution containing 1% sodium hypochlorite for 2 to 3 s, rinsed in sterile distilled water, and then cultured on potato dextrose agar (PDA) medium. Fungal colonies initially produced a white mycelium that became greenish gray when incubated at temperatures ranging between 22 and 25°C under alternating daylight and darkness (13 h of light and 11 h of dark). After 14 days of incubation, unicellular, cylindrical or truncated cone-shaped, light brown chlamydospores measuring 6 to 12 μm in diameter developed in long chains. The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS4/ITS6 and sequenced. BLAST analysis (1) of the 498-bp segment showed 100% homology with a sequence of a Phoma sp. (EF585395). The nucleotide sequence of our isolate was assigned GenBank Accession No. GU573979. Pathogenicity tests were performed by placing 100 ml of a water homogenate of mycelium (1 × 105 mycelial fragments/ml) obtained from 15-day-old PDA cultures of the fungus on leaves of three healthy 4-month-old potted R. fulgida plants. Three plants inoculated with a homogenate of PDA served as controls. Plants were maintained in a greenhouse, in a high humidity chamber for 7 days after inoculation, at temperatures ranging from 18 to 22°C and under high relative humidity conditions (70 to 90%). The first foliar lesions developed on leaves 7 days after inoculation, and after 10 to 12 days, 80% of leaves were severely infected. Control plants remained healthy. The organism reisolated on PDA from leaf lesions was identical in morphology to the isolate used for inoculation. The pathogenicity test was carried out twice. To our knowledge, this is the first report of the presence of a Phoma sp. on R. fulgida in Italy. Mycosphaerella ligulicola was reported on Rudbeckia sp. (2), while M. rudbeckiae and Phoma exigua have been reported on R. hirta (3). Currently, the economic importance of this disease is limited, but may become a more significant problem if the cultivation of this species increases. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) C. G. C. Chesters and J. P. Blakeman. Ann. Appl. Biol. 60:385, 1967. (3) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN, 1989.

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First Report of Stem and Foliage Blight of Chrysanthemum Caused by Phytophthora drechsleri in the United States

Plant Disease ◽

10.1094/pdis-03-21-0631-pdn ◽

2021 ◽

Author(s):

Charles Krasnow ◽

Nancy Rechcigl ◽

Jennifer Olson ◽

Linus Schmitz ◽

Steven N. Jeffers

Keyword(s):

United States ◽

South Carolina ◽

Sequence Similarity ◽

Morphological Characteristics ◽

The United States ◽

Universal Primers ◽

Broad Host Range ◽

Pathogenicity Test ◽

First Report ◽

Foliage Blight

Chrysanthemum (Chrysanthemum × morifolium) plants exhibiting stem and foliage blight were observed in a commercial nursery in eastern Oklahoma in June 2019. Disease symptoms were observed on ~10% of plants during a period of frequent rain and high temperatures (26-36°C). Dark brown lesions girdled the stems of symptomatic plants and leaves were wilted and necrotic. The crown and roots were asymptomatic and not discolored. A species of Phytophthora was consistently isolated from the stems of diseased plants on selective V8 agar (Lamour and Hausbeck 2000). The Phytophthora sp. produced ellipsoid to obpyriform sporangia that were non-papillate and persistent on V8 agar plugs submerged in distilled water for 8 h. Sporangia formed on long sporangiophores and measured 50.5 (45-60) × 29.8 (25-35) µm. Oospores and chlamydospores were not formed by individual isolates. Mycelium growth was present at 35°C. Isolates were tentatively identified as P. drechsleri using morphological characteristics and growth at 35°C (Erwin and Ribeiro 1996). DNA was extracted from mycelium of four isolates, and the internal transcribed spacer (ITS) region was amplified using universal primers ITS 4 and ITS 6. The PCR product was sequenced and a BLASTn search showed 100% sequence similarity to P. drechsleri (GenBank Accession Nos. KJ755118 and GU111625), a common species of Phytophthora that has been observed on ornamental and vegetable crops in the U.S. (Erwin and Ribeiro 1996). The gene sequences for each isolate were deposited in GenBank (accession Nos. MW315961, MW315962, MW315963, and MW315964). These four isolates were paired with known A1 and A2 isolates on super clarified V8 agar (Jeffers 2015), and all four were mating type A1. They also were sensitive to the fungicide mefenoxam at 100 ppm (Olson et al. 2013). To confirm pathogenicity, 4-week-old ‘Brandi Burgundy’ chrysanthemum plants were grown in 10-cm pots containing a peat potting medium. Plants (n = 7) were atomized with 1 ml of zoospore suspension containing 5 × 103 zoospores of each isolate. Control plants received sterile water. Plants were maintained at 100% RH for 24 h and then placed in a protected shade-structure where temperatures ranged from 19-32°C. All plants displayed symptoms of stem and foliage blight in 2-3 days. Symptoms that developed on infected plants were similar to those observed in the nursery. Several inoculated plants died, but stem blight, dieback, and foliar wilt were primarily observed. Disease severity averaged 50-60% on inoculated plants 15 days after inoculation. Control plants did not develop symptoms. The pathogen was consistently isolated from stems of symptomatic plants and verified as P. drechsleri based on morphology. The pathogenicity test was repeated with similar results. P. drechsleri has a broad host range (Erwin and Ribeiro 1996; Farr et al. 2021), including green beans (Phaseolus vulgaris), which are susceptible to seedling blight and pod rot in eastern Oklahoma. Previously, P. drechsleri has been reported on chrysanthemums in Argentina (Frezzi 1950), Pennsylvania (Molnar et al. 2020), and South Carolina (Camacho 2009). Chrysanthemums are widely grown in nurseries in the Midwest and other regions of the USA for local and national markets. This is the first report of P. drechsleri causing stem and foliage blight on chrysanthemum species in the United States. Identifying sources of primary inoculum may be necessary to limit economic loss from P. drechsleri.

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First Report of a Leaf Spot on Conyza sumatrensis Caused by Phoma macrostoma in China

Plant Disease ◽

10.1094/pdis-03-11-0228 ◽

2012 ◽

Vol 96 (1) ◽

pp. 148-148 ◽

Cited By ~ 1

Author(s):

J. Liu ◽

H. D. Luo ◽

W. Z. Tan ◽

L. Hu

Keyword(s):

Leaf Spot ◽

Fungal Pathogens ◽

Biological Diversity ◽

Biocontrol Agent ◽

Continuous Light ◽

The United States ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Dry Land ◽

First Report

Conyza sumatrensis (Asteraceae), an annual or biennial plant, is native to North and South America. It is an invasive, noxious weed that is widespread in southern and southeastern China. It invades farm land and causes great losses to dry land crops, including wheat, corn, and beans. It also reduces biological diversity by crowding out native plants in the infested areas (3,4). During a search for fungal pathogens that could serve as potential biological control agents of C. sumatrensis, a leaf spot disease was observed in 2010 in Chongqing, China. An isolate (SMBC22) of a highly virulent fungus was obtained from diseased leaves. Pathogenicity tests were performed by placing 6-mm-diameter mycelial disks of 7-day-old potato dextrose agar (PDA) cultures of SMBC22 on leaves of 15 healthy greenhouse-grown plants of C. sumatrensis; the same number of control plants was treated with sterile PDA disks. Treated plants were covered with plastic bags for 24 h and maintained in a growth chamber with daily average temperatures of 24 to 26°C, continuous light (3,100 lux), and high relative humidity (>90%). Lesions similar to those observed in the field were first obvious on the SMBC22-inoculated leaves 3 days after inoculation. Symptoms became severe 7 to 9 days after inoculation. Control plants remained healthy. The fungus was reisolated from inoculated and diseased leaves and it was morphologically the same as SMBC22. The pathogenicity test was conducted three times. A survey of 10 southern and southeastern Chinese provinces revealed that the disease was widespread and it attacked leaves and stems of seedlings and mature plants of C. sumatrensis. Lesions on leaves were initially small, circular, and water soaked. The typical lesion was ovoid or fusiform, dark brown, and surrounded by a yellow halo. The spots coalesced to form large lesions and plants were often completely blighted. Fungal colonies of SMBC22 on PDA plates were initially white and turned dark gray. Colonies were circular with smooth edges with obvious rings of pycnidia on the surface. Aerial hyphae were short and dense. Pycnidia, black and immersed or semi-immersed in the medium, were visible after 12 days of incubation. Pycnidia were 72 to 140 μm in diameter. Conidia were produced in the pycnidia and were hyaline, unicellular, ellipsoidal, and 4.4 to 6.1 × 1.6 to 2.2 μm. To confirm identification of the fungus, genomic DNA was extracted from mycelia of a 7-day-old culture on PDA at 25°C (2). The internal transcribed spacer (ITS) gene of rDNA was amplified using primers ITS4/ITS5. The gene sequence was 524 bp long and registered in NCBI GenBank (No. HQ645974). BLAST analysis showed that the current sequence had 99% homology to an isolate of Phoma macrostoma (DQ 404792) from Cirsium arvense (Canada thistle) in Canada and reported to cause chlorotic symptoms on that host plant (1). To our knowledge, this is the first report of P. macrostoma causing disease on C. sumatrensis in China. P. macrostoma, thought of as a biocontrol agent of broadleaf weeds in Canada, has been patented in the United States. The current isolate of P. macrostoma is considered as a potential biocontrol agent of C. sumatrensis. References: (1) P. R. Graupner et al. J. Nat. Prod. 66:1558, 2004. (2) S. Takamatsu et al. Mycoscience 42:135, 2001. (3) W. Z. Tan et al. Page 177 in: Manual of Emergency Control Technology Invasive Pests in China. G. L. Zhang, ed. Science Press, Beijing, 2010. (4) C. Wang et al. J. Wuhan Bot. Res. 28:90, 2010.

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First Report of Alternaria Leaf Spot of Banana Caused by Alternaria alternata in the United States

Plant Disease ◽

10.1094/pdis-01-13-0007-pdn ◽

2013 ◽

Vol 97 (8) ◽

pp. 1116-1116 ◽

Cited By ~ 3

Author(s):

V. Parkunan ◽

S. Li ◽

E. G. Fonsah ◽

P. Ji

Keyword(s):

United States ◽

Alternaria Alternata ◽

Leaf Spot ◽

Morphological Characteristics ◽

The United States ◽

Its Sequencing ◽

Single Spore ◽

First Report ◽

Alternaria Leaf Spot ◽

Leaf Spots

Research efforts were initiated in 2003 to identify and introduce banana (Musa spp.) cultivars suitable for production in Georgia (1). Selected cultivars have been evaluated since 2009 in Tifton Banana Garden, Tifton, GA, comprising of cold hardy, short cycle, and ornamental types. In spring and summer of 2012, 7 out of 13 cultivars (African Red, Blue Torres Island, Cacambou, Chinese Cavendish, Novaria, Raja Puri, and Veinte Cohol) showed tiny, oval (0.5 to 1.0 mm long and 0.3 to 0.9 mm wide), light to dark brown spots on the adaxial surface of the leaves. Spots were more concentrated along the midrib than the rest of the leaf and occurred on all except the newly emerged leaves. Leaf spots did not expand much in size, but the numbers approximately doubled during the season. Disease incidences on the seven cultivars ranged from 10 to 63% (10% on Blue Torres Island and 63% on Novaria), with an average of 35% when a total of 52 plants were evaluated. Six cultivars including Belle, Ice Cream, Dwarf Namwah, Kandarian, Praying Hands, and Saba did not show any spots. Tissue from infected leaves of the seven cultivars were surface sterilized with 0.5% NaOCl, plated onto potato dextrose agar (PDA) media and incubated at 25°C in the dark for 5 days. The plates were then incubated at room temperature (23 ± 2°C) under a 12-hour photoperiod for 3 days. Grayish black colonies developed from all the samples, which were further identified as Alternaria spp. based on the dark, brown, obclavate to obpyriform catenulate conidia with longitudinal and transverse septa tapering to a prominent beak attached in chains on a simple and short conidiophore (2). Conidia were 23 to 73 μm long and 15 to 35 μm wide, with a beak length of 5 to 10 μm, and had 3 to 6 transverse and 0 to 5 longitudinal septa. Single spore cultures of four isolates from four different cultivars were obtained and genomic DNA was extracted and the internal transcribed spacer (ITS1-5.8S-ITS2) regions of rDNA (562 bp) were amplified and sequenced with primers ITS1 and ITS4. MegaBLAST analysis of the four sequences showed that they were 100% identical to two Alternaria alternata isolates (GQ916545 and GQ169766). ITS sequence of a representative isolate VCT1FT1 from cv. Veinte Cohol was submitted to GenBank (JX985742). Pathogenicity assay was conducted using 1-month-old banana plants (cv. Veinte Cohol) grown in pots under greenhouse conditions (25 to 27°C). Three plants were spray inoculated with the isolate VCT1FT1 (100 ml suspension per plant containing 105 spores per ml) and incubated under 100% humidity for 2 days and then kept in the greenhouse. Three plants sprayed with water were used as a control. Leaf spots identical to those observed in the field were developed in a week on the inoculated plants but not on the non-inoculated control. The fungus was reisolated from the inoculated plants and the identity was confirmed by morphological characteristics and ITS sequencing. To our knowledge, this is the first report of Alternaria leaf spot caused by A. alternata on banana in the United States. Occurrence of the disease on some banana cultivars in Georgia provides useful information to potential producers, and the cultivars that were observed to be resistant to the disease may be more suitable for production. References: (1) E. G. Fonsah et al. J. Food Distrib. Res. 37:2, 2006. (2) E. G. Simmons. Alternaria: An identification manual. CBS Fungal Biodiversity Center, Utrecht, Netherlands, 2007.

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First Report of Ramularia coleosporii causing Leaf Spot on Perilla frutescens in Korea

Plant Disease ◽

10.1094/pdis-01-21-0223-pdn ◽

2021 ◽

Author(s):

Md Aktaruzzaman ◽

Tania Afroz ◽

Hyo-Won Choi ◽

Byung Sup Kim

Keyword(s):

Leaf Spot ◽

Ipomoea Batatas ◽

Rust Fungus ◽

Morphological Characteristics ◽

Perilla Frutescens ◽

Herbaceous Plant ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Single Spore ◽

First Report

Perilla (Perilla frutescens var. japonica), a member of the family Labiatae, is an annual herbaceous plant native to Asia. Its fresh leaves are directly consumed and its seeds are used for cooking oil. In July 2018, leaf spots symptoms were observed in an experimental field at Gangneung-Wonju National University, Gangneung, Gangwon province, Korea. Approximately 30% of the perilla plants growing in an area of about 0.1 ha were affected. Small, circular to oval, necrotic spots with yellow borders were scattered across upper leaves. Masses of white spores were observed on the leaf underside. Ten small pieces of tissue were removed from the lesion margins of the lesions, surface disinfected with NaOCl (1% v/v) for 30 s, and then rinsed three times with distilled water for 60 s. The tissue pieces were then placed on potato dextrose agar (PDA) and incubated at 25°C for 7 days. Five single spore isolates were obtained and cultured on PDA. The fungus was slow-growing and produced 30-50 mm diameter, whitish colonies on PDA when incubated at 25ºC for 15 days. Conidia (n= 50) ranged from 5.5 to 21.3 × 3.5 to 5.8 μm, were catenate, in simple or branched chains, ellipsoid-ovoid, fusiform, and old conidia sometimes had 1 to 3 conspicuous hila. Conidiophores (n= 10) were 21.3 to 125.8 × 1.3 to 3.6 μm in size, unbranched, straight or flexuous, and hyaline. The morphological characteristics of five isolates were similar. Morphological characteristics were consistent with those described for Ramularia coleosporii (Braun, 1998). Two representative isolates (PLS 001 & PLS003) were deposited in the Korean Agricultural Culture Collection (KACC48670 & KACC 48671). For molecular identification, a multi-locus sequence analysis was conducted. The internal transcribed spacer (ITS) regions of the rDNA, partial actin (ACT) gene and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene were amplified using primer sets ITS1/4, ACT-512F/ACT-783R and gpd1/gpd2, respectively (Videira et al. 2016). Sequences obtained from each of the three loci for isolate PLS001 and PLS003 were deposited in GenBank with accession numbers MH974744, MW470869 (ITS); MW470867, MW470870 (ACT); and MW470868, MW470871 (GAPDH), respectively. Sequences for all three genes exhibited 100% identity with R. coleosporii, GenBank accession nos. GU214692 (ITS), KX287643 (ACT), and 288200 (GAPDH) for both isolates. A multi-locus phylogenetic tree, constructed by the neighbor-joining method with closely related reference sequences downloaded from the GenBank database and these two isolates demonstrated alignment with R. coleosporii. To confirm pathogenicity, 150 mL of a conidial suspension (2 × 105 spores per mL) was sprayed on five, 45 days old perilla plants. An additional five plants, to serve as controls, were sprayed with sterile water. All plants were placed in a humidity chamber (>90% relative humidity) at 25°C for 48 h after inoculation and then placed in a greenhouse at 22/28°C (night/day). After 15 days leaf spot symptoms, similar to the original symptoms, developed on the leaves of the inoculated plants, whereas the control plants remained symptomless. The pathogenicity test was repeated twice with similar results. A fungus was re-isolated from the leaf lesions on the inoculated plants which exhibited the same morphological characteristics as the original isolates, fulfilling Koch’s postulates. R. coleosporii has been reported as a hyperparasite on the rust fungus Coleosporium plumeriae in India & Thailand and also as a pathogen infecting leaves of Campanula rapunculoides in Armenia, Clematis gouriana in Taiwan, Ipomoea batatas in Puerto Rico, and Perilla frutescens var. acuta in China (Baiswar et al. 2015; Farr and Rossman 2021). To the best of our knowledge, this is the first report of R. coleosporii causing leaf spot on P. frutescens var. japonica in Korea. This disease poses a threat to production and management strategies to minimize leaf spot should be developed.

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First Report of Nigrospora osmanthi Causing Leaf Spot on Tartary Buckwheat in China

Plant Disease ◽

10.1094/pdis-08-20-1773-pdn ◽

2020 ◽

Author(s):

Quan Shen ◽

Xixu Peng ◽

Feng He ◽

Shaoqing Li ◽

Zuyin Xiao ◽

...

Keyword(s):

Relative Humidity ◽

Leaf Spot ◽

Plant Pathogens ◽

Hunan Province ◽

Tartary Buckwheat ◽

Tween 20 ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Sterile Water ◽

First Report

Buckwheat (Fagopyrum tataricum) is a traditional short-season pseudocereal crop originating in southwest China and is cultivated around the world. Antioxidative substances in buckwheat have been shown to provide many potential cardiovascular health benefits. Between August and November in 2019, a leaf spot was found in several Tartary buckwheat cv. Pinku1 fields in Xiangxiang County, Hunan Province, China. The disease occurred throughout the growth cycle of buckwheat after leaves emerged, and disease incidence was approximately 50 to 60%. Initially infected leaves developed a few round lesions, light yellow to light brown spots. Several days later, lesions began to enlarge with reddish brown borders, and eventually withered and fell off. Thirty lesions (2×2 mm) collected from three locations with ten leaves in each location were sterilized in 70% ethanol for 10 sec, in 2% sodium hypochlorite for 30 sec, rinsed in sterile water for three times, dried on sterilized filter paper, and placed on a potato dextrose PDA with lactic acid (3 ml/L), and incubated at 28°C in the dark for 3 to 5 days. Fungal colonies were initially white and later turned black with the onset ofsporulation. Conidia were single-celled, black, smooth, spherical to subspherical, and measured 9.2 to 15.6 µm long, and 7.1 to 11.6 µm wide (n=30). Each conidium was terminal and borne on a hyaline vesicle at the tip of conidiophores. Morphologically, the fungus was identified as Nigrospora osmanthi (Wang et al. 2017). Identification was confirmed by amplifying and sequencing the ITS region, and translation elongation factor 1-alpha (TEF1-α) and partial beta-tublin (TUB2) genes using primers ITS1/ITS4 (Mills et al. 1992), EF1-728F/EF-2 (Carbone and Kohn 1999; O’Donnell et al. 1998) and Bt-2a/Bt-2b (Glass et al. 1995), respectively. BLAST searches in GenBank indicated the ITS (MT860338), TUB2 (MT882054) and TEF1-α (MT882055) sequences had 99.80%, 99% and 100% similarity to sequences KX986010.1, KY019461.1 and KY019421.1 of Nigrospora osmanthi ex-type strain CGMCC 3.18126, respectively. A neighbor-joining phylogenetic tree constructed using MEGA7.0 with 1,000 bootstraps based on the concatenated nucleotide sequences of the three genes indicated that our isolate was closely related to N. osmanthi. Pathogenicity test was performed using leaves of healthy F. tataricum plants. The conidial suspension (1 × 106 conidia/ml) collected from PDA cultures with 0.05% Tween 20 buffer was used for inoculation by spraying leaves of potted 20-day-old Tartary buckwheat cv. Pinku1. Five leaves of each plant were inoculated with spore suspensions (1 ml per leaf). An equal number of control leaves were sprayed with sterile water to serve as a control. The treated plants were kept in a greenhouse at 28°C and 80% relative humidity for 24 h, and then transferred to natural conditions with temperature ranging from 22 to 30°C and relative humidity ranging from 50 to 60%. Five days later, all N. osmanthi-inoculated leaves developed leaf spot symptoms similar to those observed in the field, whereas control leaves remained healthy. N. osmanthi was re-isolated from twelve infected leaves with frequency of 100%, fulfilling Koch’s postulates. The genus Nigrospora has been regarded by many scholars as plant pathogens (Fukushima et al. 1998) and N. osmanthi is a known leaf blight pathogen for Stenotaphrum secundatum (Mei et al. 2019) and Ficus pandurata (Liu et al. 2019) but has not been reported on F. tataricum. Nigrospora sphaerica was also detected in vegetative buds of healthy Fagopyrum esculentum Moench (Jain et al. 2012). To our knowledge, this is the first report of N. osmanthi causing leaf spot on F. tataricum in China and worldwide. Appropriate strategies should be developed to manage this disease.

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First Report of Wilt of Rubber Tree Caused by Chalaropsis thielavioides in China

Plant Disease ◽

10.1094/pdis-09-20-2066-pdn ◽

2020 ◽

Author(s):

Xue Li ◽

Jie Li ◽

Hua Yong Bai ◽

Kecheng Xu ◽

Ruiqi Zhang ◽

...

Keyword(s):

Rubber Tree ◽

Morphological Characteristics ◽

Spore Suspension ◽

Malt Extract ◽

Pathogenicity Test ◽

Distilled Water ◽

Ceratocystis Fimbriata ◽

First Report ◽

Mould Fungus ◽

Its Sequence Analysis

Rubber tree (Hevea brasiliensis (Willd. ex Adr. Juss) Müll. Arg.) is used for the extraction of natural rubber and is an economically and socially important estate crop commodity in many Asian countries such as Indonesia, Malaysia, Thailand, India, Sri Lanka, China and several countries in Africa (Pu et al, 2007). Xishuangbanna City and Wenshan City are the main rubber cultivation areas in Yunnan Province, China. In November 2012, rubber tree showing typical wilt symptoms (Fig. 1 A) and vascular stains (Fig. 1 B) were found in Mengla County, Xishuangbanna City. This disease was destructive in these trees and plant wilt death rate reached 5%. The diseased wood pieces (0.5cm long) from trunk of rubber was surface disinfected with 75% ethanol for 30s and 0.1% mercuric chloride (HgCl2) for 2min, rinsed three times with sterile distilled water, plated onto malt extract agar medium (MEA), and incubated at 28℃. After 7 days, fungal-like filaments were growing from the diseased trunk. Six cultures from 6 rubber trunk were obtained and incubated on MEA at 28℃, after 7 days to observe the cultural features. The mycelium of each culture was white initially on MEA, and then became dark green. Cylindrical endoconidia apices rounded, non-septate, smooth, single or borne in chains (8.9 to 23.6 × 3.81 to 6.3μm) (Fig. 1 C). Chlamydospores (Fig. 1 D) were abundant, thick walled, smooth, forming singly or in chains (11.1 to 19.2 × 9.4 to 12.0μm). The mould fungus was identifed as Chalaropsis based on morphology (Paulin-Mahady et al. 2002). PCR amplification was carried out for 3 isolates, using rDNA internal transcribed spacer (ITS) primer pairs ITS1F and ITS4 (Thorpe et al. 2005). The nucleotide sequences were deposited in the GenBank data base and used in a Blast search of GenBank. Blast analysis of sequenced isolates XJm8-2-6, XJm8-2 and XJm10-2-6 (accessions KJ511486, KJ511487, KJ511489 respectively) had 99% identity to Ch. thielavioides strains hy (KF356186) and C1630 (AF275491). Thus the pathogen was identified as Ch. thielavioides based on morphological characteristics and rDNA-ITS sequence analysis. Pathogenicity test of the isolate (XJm8-2) was conducted on five 1-year-old rubber seedlings. The soil of 5 rubber seedlings was inoculated by drenching with 40 ml spore suspension (106 spores / ml). Five control seedlings were inoculated with 40 ml of sterile distilled water. All the seedlings were maintained in a controlled greenhouse at 25°C and watered weekly. After inoculated 6 weeks, all the seedlings with spore suspension produced wilt symptoms, as disease progressed, inoculated leaves withered (Fig. 1 E) and vascular stains (Fig. 1 F) by 4 months. While control seedlings inoculated with sterile distilled water remained healthy. The pathogen re-isolated from all inoculated symptomatic trunk was identical to the isolates by morphology and ITS analysis. But no pathogen was isolated from the control seedlings. The pathogenicity assay showed that Ch. thielavioides was pathogenic to rubber trees. Blight caused on rubber tree by Ceratocystis fimbriata previously in Brazil (Valdetaro et al. 2015), and wilt by Ch. thielavioides was not reported. The asexual states of most species in Ceratocystis are “chalara” or “thielaviopsis” (de Beer et al. 2014). To our knowledge, this is the first report of this fungus causing wilt of rubber in China. The spread of this disease may pose a threat to rubber production in China.

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First Report of Leaf Blight on Fan Columbine (Aquilegia flabellata) Caused by Rhizoctonia solani AG 4 in Italy

Plant Disease ◽

10.1094/pdis-93-4-0433a ◽

2009 ◽

Vol 93 (4) ◽

pp. 433-433 ◽

Cited By ~ 2

Author(s):

A. Garibaldi ◽

G. Gilardi ◽

D. Bertetti ◽

M. L. Gullino

Keyword(s):

United States ◽

Rhizoctonia Solani ◽

Aqueous Suspension ◽

Morphological Characteristics ◽

The United States ◽

Leaf Blight ◽

Herbaceous Plant ◽

Pathogenicity Test ◽

First Report ◽

Leaf Petiole

Aquilegia flabellata (Ranunculaceae), fan columbine, is a perennial herbaceous plant with brilliant blue-purple flowers with white petal tips. It can also be grown for cut flower production. In April of 2008, in several nurseries located near Biella (northern Italy), a leaf blight was observed on 10 to 15% of potted 30-day-old plants grown on a sphagnum peat substrate at 15 to 20°C and relative humidity of 80 to 90%. Semicircular, water-soaked lesions developed on leaves just above the soil line at the leaf-petiole junction and later along the leaf margins. Lesions expanded over several days along the midvein until the entire leaf was destroyed. Blighted leaves turned brown, withered, and abscised. Severely infected plants died. Diseased tissue was disinfested for 10 s in 1% NaOCl, rinsed with sterile water, and plated on potato dextrose agar (PDA) amended with 25 mg/liter streptomycin sulfate. A fungus with the morphological characteristics of Rhizoctonia solani was consistently recovered, then transferred and maintained in pure culture. Ten-day-old mycelium grown on PDA at 22 ± 1°C appeared light brown, rather compact, and had radial growth. Sclerotia were not present. Isolates obtained from affected plants successfully anastomosed with tester isolate AG 4 (AG 4 RT 31, obtained from tobacco plants). Results were consistent with other reports on anastomosis reactions (2). Pairings were also made with tester isolates of AG 1, 2.1, 2.2, 3, 6, 7, 11, and BI with no anastomoses observed between the recovered and tester isolates. The internal transcribed spacer (ITS) region of rDNA was amplified using primers ITS4/ITS6 and sequenced. BLASTn analysis (1) of the 648-bp fragment showed a 100% homology with the sequence of R. solani AG-4 AB000018. The nucleotide sequence has been assigned GenBank Accession No. FJ 534555. For pathogenicity tests, the inoculum of R. solani was prepared by growing the pathogen on PDA for 10 days. Five plants of 30-day-old A. flabellata were grown in 3-liter pots. Inoculum consisting of an aqueous suspension of PDA and mycelium disks (5 g of mycelium + agar per plant) was placed at the collar of plants. Five plants inoculated with water and PDA fragments alone served as control treatments. Plants were maintained in a greenhouse at temperatures between 20 and 24°C. The first symptoms, similar to those observed in the nursery, developed 7 days after the artificial inoculation. R. solani was consistently reisolated from infected leaves and stems. Control plants remained healthy. The pathogenicity test was carried out twice with similar results. The presence of R. solani AG1-IB on A. flabellata has been reported in Japan (4), while in the United States, Rhizoctonia sp. is described on Aquilegia sp. (3). This is, to our knowledge, the first report of leaf blight of A. flabellata caused by R. solani in Italy as well as in Europe. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) D. E. Carling. Grouping in Rhizoctonia solani by hyphal anastomosis reactions. In: Rhizoctonia Species: Taxonomy, Molecular Biology, Ecology, Pathology and Disease Control. Kluwer Academic Publishers, The Netherlands, 1996. (3) D. F. Farr et al. Fungi on Plants and Products in the United States. The American Phytopathological Society, St Paul, MN, 1989. (4) E. Imaizumi et al. J. Gen. Plant Pathol. 66:210, 2000.

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First report of leaf spot disease caused by Stemphylium eturmiunum on garlic in Korea.

Plant Disease ◽

10.1094/pdis-03-21-0674-pdn ◽

2021 ◽

Author(s):

Walftor Dumin ◽

Mi-Jeong Park ◽

You-Kyoung Han ◽

Yeong-Seok Bae ◽

Jong-Han Park ◽

...

Keyword(s):

Leaf Spot ◽

Allium Sativum ◽

Morphological Characteristics ◽

Fungal Isolate ◽

Causal Agent ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

First Report ◽

Spot Disease ◽

Intact Leaves

Garlic (Allium sativum L. cv.namdo) is one of the most popular vegetables grown in Korea due to its high demand from the food industry. However, garlic is susceptible to a wide range of pest infestations and diseases that cause a significant decrease in garlic production, locally and globally (Schwartz and Mohan 2008). In early 2019, the occurrence of leaf blight disease was found spreading in garlic cultivation areas around Jeonnam (34.9671107, 126.4531825) province, Korea. Disease occurrence was estimated to affect 20% of the garlic plants and resulted in up to a 3-5% decrease in its total production. At the early stage of infection, disease symptoms were manifested as small, white-greyish spots with the occurrence of apical necrosis on garlic leaves. This necrosis was observed to enlarge, producing a water-soaked lesion before turning into a black-violet due to the formation of conidia. As the disease progressed, the infected leaves wilted, and the whole garlic plants eventually died. To identify the causal agent, symptomatic tissues (brown dried water-soak lesion) were excised, surface sterilized with 1% NaOCl and placed on the Potato Dextrose Agar (PDA) followed by incubation at 25°C in the dark for 5 days. Among ten fungal isolates obtained, four were selected for further analyses. On PDA, fungal colonies were initially greyish white in colour but gradually turned to yellowish-brown after 15 days due to the formation of yellow pigments. Conidia were muriform, brown in colour, oblong (almost round) with an average size of 18 – 22 × 16 – 20 μm (n = 50) and possessed 6 - 8 transverse septa. Fungal mycelia were branched, septate, and with smooth-walled hyphae. Morphological characteristics described above were consistent with the morphology of Stemphylium eturmiunum as reported by Simmons (Simmons, 2001). For molecular identification, molecular markers i.e. internal transcribed spacer (ITS) and calmodulin (cmdA) genes from the selected isolates were amplified and sequenced (White et al., 1990; Carbone and Kohn 1999). Alignment analysis shows that ITS and cmdA genes sequence is 100% identical among the four selected isolates. Therefore, representative isolate i.e. NIHHS 19-142 (KCTC56750) was selected for further analysis. BLASTN analysis showed that ITS (MW800165) and cmdA (LC601938) sequences of the representative isolates were 100% identical (523/523 bp and 410/410 bp) to the reference genes in Stemphylium eturmiunum isolated from Allium sativum in India (KU850545, KU850835) respectively (Woudenberg et al. 2017). Phylogenetic analysis of the concatenated sequence of ITS and cmdA genes confirmed NIHHS 19-142 isolates is Stemphylium eturmiunum. Pathogenicity test was performed using fungal isolate representative, NIHHS 19-142. Conidia suspension (1 × 106 conidia/µL) of the fungal isolate was inoculated on intact garlic leaves (two leaves from ten different individual plants were inoculated) and bulbs (ten bulbs were used) respectively. Inoculation on intact leaves was performed at NIHHS trial farm whereas inoculated bulbs were kept in the closed container to maintain humidity above 90% and incubated in the incubator chamber at 25°C. Result show that the formation of water-soaked symptoms at the inoculated site was observed at 14 dpi on intact leaves whereas 11 dpi on bulbs. As a control, conidia suspension was replaced with sterile water and the result shows no symptoms were observed on the control leaves and bulbs respectively. Re-identification of fungal colonies from symptomatic leaf and bulb was attempted. Result showed that the morphological characteristics and molecular marker sequences of the three colonies selected were identical to the original isolates thus fulfilled Koch’s postulates. Early identification of Stemphylium eturmiunum as a causal agent to leaf spot disease is crucial information to employ effective disease management strategies or agrochemical applications to control disease outbreaks in the field. Although Stemphylium eturmiunum has been reported to cause leaf spot of garlic disease in China, France and India (Woudenberg et al. 2017), to our knowledge, this is the first report of causing leaf spot disease on garlic in Korea.

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