Report Of Leaf Spot Research Articles

First Report of Leaf spot Caused by Xylaria arbuscula on Flue-cured Tobacco in China.

Flue-cured tobacco (Nicotiana tabacum L.) is a leafy, annual, solanaceous plant grown commercially for its leaves in China. Around 70% of tobacco production in China occurs in southwest China. In summer of 2019, leaf spot symptoms were observed on ten to twenty percent of tobacco plants in a 2 ha commercial field of Bijie (27.32° N, 105.29° E), Guizhou province, China. The leaf spots were white with dark-brown in edges, irregularly round and oval, and diseased tissue dropped out leaving the leaves ragged in appearance (Fig. 1A, 1B). One diseased leaf from each of five plants was sampled. From five leaves, a total of 15 small (5 mm × 5 mm) pieces of leaf tissue were cut from the edge of the lesions after surface sterilization and placed on potato dextrose agar (PDA) medium. Five fungal colonies that were similar in appearance were isolated and one was purified, BEZ22, was selected arbitrarily for identification. Mycelia of the pathogen was initally white and dense, and then black carbonized mycelia appeared from the center of the colony 7 days' after incubation. Mycelia was white, sparse and radiated when incubated on OA (oatmeal agar) (Fig. 1E, 1F, 1G, 1H). Genomic DNA of the isolate was extracted. The internal transcribed spacers (ITS) with primers ITS1/ITS4 (White et al. 1990), actin (ACT) gene with primers ACT-512F/ACT-738R (Hsieh et al. 2005), beta-tubulin (TUB2) with primers T1/T22 (O'Donnell & Cigelnik 1997) and RNA polymerase II second largest subunit gene (RPB2) with primers fRPB2-5F/ fRPB2-7cR (Liu et al. 1999) were amplified and sequenced, respectively. The generated sequences were deposited in GenBank with accession numbers MT804353 (ITS), MT809582 (ACT), MT799790 (TUB2) and MT799789 (RPB2). Using BLASTN searches, the sequences of each gene above were aligned with the voucher specimum, Xylaria arbuscula 89041211. The number of nucleotides that were similar for ITS (GU300090) was 550/551 (99%); for ACT (GQ421286), 266/266 bp (100%); for TUB2 (GQ478226), 1501/1501 bp (100%); and for RPB2 (GQ844805), 1135/1135 bp (100%), respectively (Fig. 2). A phylogenetic tree was constructed based on these four sequences with a final alignment of 3456 characters (ITS 551, ACT 266, TUB2 1501 and RPB2 1138). Thus, based on morphological and phylogenetic analyses, the isolate BEZ22 was identified as Xylaria arbuscula. To verify pathogenicity, six tobacco plants at seedling stage (5-6 leaves) without visible disease were inoculated using mycelial plugs (5 mm in diameter). Leaves inoculated with PDA only plugs served as controls. After inoculation, all tobacco plants were maintained in a greenhouse with 85% relative humidity at 25 oC under a 12/12 h light/dark cycle. Five days after inoculation, typical early symptoms were observed on the inoculated leaves, and not on the control leaves. Koch's postulates were fulfilled by re-isolation of the pathogen from diseased leaves. Xylaria arbuscula has also been reported as a pathogen of Macadamia in Hawaii (Wenhsiung et al. 2009) and sugarcane in Indonesia (Maryono et al. 2020). However, to our best knowledge, this is the first report of X. arbuscula causing leaf spot on tobacco in China. This leaf spot has the potential to cause serious damage to tobacco in this region that could result in reduced production, consequently disease management of this pathogen should be considered.

First report of leaf spot of bottle gourd (Lagenaria siceraria) caused by Fusarium equiseti in Pakistan

First report of leaf spot of bottle gourd (Lagenaria siceraria) caused by Fusarium equiseti in Pakistan

First Report of Leaf Spot Caused by Septoria villarsiae on Nymphoides peltata in the United States

Nymphoides peltata, commonly known as yellow floating heart, is a freshwater aquatic plant with floating leaves. It is a highly invasive aquatic weed that has been introduced into several countries, including Ireland, New Zealand, Sweden, and the United States. In September 2019, N. peltata plants exhibiting leaf spots were collected from a private pond near Buxton, York County, Maine. Leaf spots were present on a majority of plants, and pycnidia were observed in the center of the spots. Individual pycnidia were aseptically transferred to 1/4-strength potato dextrose agar. Dark gray to black slow-growing colonies were observed between 7 and 14 days. Based on the morphological characteristics, the fungus was identified as Septoria sp. Translation elongation factor 1-alpha gene was amplified, and a 570-bp sequence resulted in 100 and 99.74% homology with Septoria villarsiae strains CBS565.88 and CBS514.78 isolated from N. peltata in the Netherlands, respectively. Previously, S. villarsiae has been reported on Limnanthemum nymphoides from India and on N. peltata from Korea, Poland, Romania, and the Netherlands. To our knowledge, this is the first report of leaf spot caused by S. villarsiae on N. peltata in the United States.

First Report of Leaf Spot Caused by Fusarium equiseti on Switchgrass in Shandong, China

First Report of Leaf Spot Caused by <i>Fusarium equiseti</i> on Switchgrass in Shandong, China

First Report of Leaf Spots Caused by Alternaria alternata on Abelmoschus manihot in Italy

First Report of Leaf Spots Caused by <i>Alternaria alternata</i> on <i>Abelmoschus manihot</i> in Italy

First Report of Leaf Spots on Oat Caused by Alternaria alternata in South Korea.

Oat, a gluten-free source of vitamins, minerals, fiber, and antioxidants required for human health (Rasane et al., 2015), is increasingly being cultivated in South Korea (Ju et al. 2011). In May 2020, ~30 % Avena sativa (cv. Samhan) leaves of a commercial oat field in Jeongeup, Korea (35.3859°N, 126.5607°E), displayed a leaf spot disease at the tillering growth stage. Small, brown spots appeared initially on the lower leaves, but progressed to dark-brown oval or irregular, necrotic spots with a light-yellowish border. To isolate the pathogen, tissue excised from growing edges of about 50 lesions was surface sterilized (immersion in 70 % ethanol then 1 % sodium hypochlorite), rinsed and transferred to potato dextrose agar. Five isolates were purified using the single-spore culture method on water agar plates (Choi et al. 1999). Conidia from the PDA cultures (n = 50) were elongated, ellipsoidal, and obclavate, with one to five septa (primarily transverse), 18 to 35 μm × 7 to 12 μm. The morphological characteristics of the fungus from each culture were consistent with those observed for Alternaria alternata (Chen et al. 2020; Simmons 2007). To confirm the identity of the fungal pathogen, the internal transcribed spacer (ITS) region was amplified with universal primers ITS1/ITS4 and sequenced (White et al. 1990). BLAST analysis of the resulting nucleotide sequence of one representative isolate, Oaa-2 (GenBank MT786424), showed 100 % identity with A. alternata-type strains, such as CBS916.96. Further, nine nuclear protein-coding genes from the Oaa-2 isolate were amplified using previously described primers (Lawrence et al. 2013; Wouderengerg et al. 2015). These sequences were deposited under the following GenBank accession numbers: MT795660 (rpb2), MT795656 (His), MT795657 (CML), MT795658 (ACT), MT787343 (CHSD), MT795655 (GAPDH), MT795661 (endoPG), and MT795662 (TEF1). The nine sequences of the Oaa-2 isolate showed 99%-100 % identity to those of A. alternata-type strain CBS916.96 in NCBI. Fig. S2 shows the phylogenetic trees for the ITS and GAPDH sequences indicating high homology. Pathogenicity of this isolate was confirmed by spraying leaves of surface-sterilized, glasshouse-grown, "Samhan" oats with suspensions (5 × 106 spores/ml) of Oaa-2 conidia. Experimental seedlings were grown for 2 weeks in a chamber at 20 ± 3 °C in 60 % relative humidity with a 10-h light/14-h dark cycle (200 µmol/m2/s). Conidial suspensions were prepared for inoculation by flooding with sterile water, followed by filtering the suspension through three layers of sterile cheesecloth to remove debris. Three pots each containing twenty oat seedlings were sprayed with the Oaa-2 isolate conidial suspension (5 × 106 conidia/ml) and incubated for 10 days at 20 ± 3 °C in 100 % humidity with a 10-h photoperiod. Three healthy seedlings were sprayed with sterile water as a control. After 10 days, dark brown spots developed in the inoculated plants; however, no spots were observed in the controls. Fungi with the same conidial characteristics as Oaa-2 were isolated from the infected plants, and the ITS sequence and the nine nuclear protein-encoding genes of these plant isolates was identical to those of Oaa-2. Pathogenicity tests also were conducted with isolate Oaa-2 with two oat cultivars, Sunyang and Joyang, with the same result. To our knowledge, A. alternata has not been previously reported to cause disease on oats in South Korea, although it does cause economic losses in China (Chen et al. 2020).

First Report of Leaf Spot on Akebia trifoliata Caused by Phytophthora nicotianae in China.

Akebia trifoliata (Thunb.) Koidz. is a species in the family Lardizabalaceae, which belongs to deciduous woody lianas. It is an important species of plant used in Chinese medicine. In July 2019, a leaf spot disease was observed on A. trifoliata in a nursery garden in Jingzhou (N 30° 21', E 112° 19'), Hubei Province, China. Symptoms initially appeared as small brown spots and subsequently developed into subcircular or irregular-shaped brown necrotic lesions. In severe cases, the leaves became completely necrotic and abscised. The incidence of leaf symptoms on affected plants ranged was between 30% and 40%. To isolate the pathogen, pieces of symptomatic leaves were collected and excised at the margins of lesions, surface disinfected with 70% ethanol and 0.1% HgCl2, rinsed three times with sterile water, placed on potato dextrose agar (PDA) amended with 50 μg/ml kanamycin, and incubated at 28°C in the dark for 3 days. Isolated colonies were subcultured by transferring hyphal tips. Six fungal isolates were isolated from the collected tissues. All six isolates had similar colony morphologies on on PDA and were composed of white flocculent aerial hyphae. The average radial growth rate of colonies after 7 days was 11.2 mm/d. Isolates were later cultured on 20% V8 juice agar for 20 days to encourage sporulation. Sporangia were produced on V8 media and were colorless, inverted, pear-shaped, and terminal, with obvious mastoid, 22 to 34 × 28 to 46µm (n=50); Oospores were light brown, and suborbicular, with thick wall, 18 to 26µm (n=20); Globose chlamydospores were light brown, and suborbicular, 12 to 32µm (n=50). Antheridia were not observed suggesting homothallism. These morphological charactertistics were identical to those reported for Phytophthora nicotianae (Erwin and Ribeiro 1996). We selected a single isolate 'B2', for molecular identification because it was the most aggressive in leaf pathogenicity assays. The internal transcribed spacer (ITS) region of rDNA was amplified and sequenced using primers ITS1/ITS4 (White et al. 1990). BLAST analyis revealed that the ITS sequence (GenBank accession nos. MT472132) was 100% identical to other P. nicotianae strains (GenBank accession nos. KJ754387). To fulfill Koch's postulates, a 50 ml zoospores suspension (106 spores/ml) of B2 was sprayed on the foliage of three 1-year-old healthy seedlings. Sterile distilled water to inoculate control plants. After 10 days, typical symptoms of dark brown spots were observed on all the inoculated leaves, while the control leaves remained asymptomatic. P. nicotianae was re-isolated from the inoculated, symptomatic leaves, thus confirming Koch's hypothesis. The experiment was repeated three times. To the best of our knowledge, this is the first report of P. nicotianae causing leaf spot on A. trifoliata in China. P. nicotianae is a common stramenopile pathogen that infects many plant hosts. The presence of this pathogen in an A. trifoliata nursery should be carefully considered to mitigate possible outbreaks of this disease in other fields in this growing region.

First Report of Corynspora cassiicola Causing Leaf Spot on Jasminum sambac in China.

Jasminum sambac (Linnaeus) Aiton belonging to Oleaceae family, has been cultivated in China over 1500 years. It is an important tea crop in South China, and popular ornamental plant in North China. In June 2015, Corynespora leaf spot on Jasminum sambac was first observed in Caozhuang in Tianjin(E: 117°20', N: 39°13'). In a series of investigations, the disease incidence of Corynespora leaf spot on Jasminum sambac in Tianjin was found to be as high as 20%. The disease was also found in flower planting greenhouses in Beijing and Hebei region. The initial symptoms appeared as small circular spot with gray white in the center and dark brown in the edge. The lesions expanded rapidly to 5-10 mm diameter, surrounded by a yellow halo. The affected leaves became covered with the circular spots and defoliated eventually. Fifty-six leaf samples were collected from 20 greenhouses, then directly examined and disease tissues set aside for isolation. Conidiophores were either solitary or in clusters, straight or curved and un-branched, with 3-10 septate, pale to dark brown in color. Those were measured 80.6 to 234.8 μm long and 4.5 to 8.0 μm wide in size and were formed with 0-8 successive cylindrical proliferation (n = 50). Conidia borne from conidiophore, were single or in chains, obclavate to cylindrical, smooth. The average size of 50 conidia was 43.6 to 130.7 μm long×6.4 to 13.5 μm wide, with 4 to 8 pseudosepta (n = 50). The infected leaves were cut into small pieces, sterilized with 75% ethanol, rinsed in sterilized distilled water and then plated on potato dextrose agar (PDA). The isolated plates were incubated at 25℃ for 5 days. Thrity-five pure culture isolates were obtained from diseased leaves. The colonies were pale brown on the front and dark brown on the underneath, with thickly aerial mycelia. Morphological characteristics of the pathogen from the infected leaves and purified strains matched descriptions of Corynesora cassicola (Ellis and Holliday 1971). To confirm the pathogenicity, conidial suspensions (106 conidia mL-1 ) of 35 isolated strains were sprayed on the healthy plants at the seedling stage of Jasminum sambac respectively, 5 plants per strains. 10 healthy plants were sprayed only with sterilized water as control. Conidial suspensions were prepared by washing conidia from the pure C. cassiicola cultures. Conidial suspensions were adjusted to a concentration of 1 × 106 conidia/mL with the hemocytometer. All plants were incubated in artificial climate-room at 90% relative humidity, 25±2℃. After 7 days, small circular spots, similar to the field samples were observed on all plants inoculated with the pathogen. By comparison, control plants displayed no symptoms. The same pathogens were re-isolated from the symptomatic leaves fulfilling Koch's postulates. For molecular identification, rDNA-ITS and two housekeeping genes (TUB2 and EF-1α) gene were amplified and sequenced (Zhu et al. 2020). The sequences of three high pathogenicity isolats showed 99% homology to C.cassiicola (MK571399) with464 bp for rDNA-ITS, 100% homology to C.cassiicola (MN512639) with469 bp for TUB2 gene, 98.61% homology to C.cassiicola (MH263735) with287 bp for EF-1α gene. And all 9 sequences were submitted to GenBank (GenBank accession No. MT334575-77 for rDNA-ITS, MT886387-89 for TUB2, and MT886390-92 for EF-1α).The molecular identification and morphological characteristics showed that the isolated strains from Jasminum sambac were identified as C.cassiicola. C. cassiicola has an extensive host range, covering 397 host plants (Farr and Rossman 2020). Recently, C.cassiicola has been reported on Jasminum mesnyi of Oleaceaein China (Zhang et al. 2018). To our knowledge, this is the first report of leaf spot caused by C.cassiicola on Jasminum sambac in China and also worlwide.

First Report of Leaf Spot Caused by Alternaria alternata on Phlox maculata in Italy

Phlox maculata (common name spotted phlox), Polemoniaceae family, is an herbaceous perennial plant with showy panicle inflorescences, commonly grown in mixed borders. During summer 2019, about twenty 5-year-old plants of P. maculata growing in a garden located in the Biella province (northern Italy) showed symptoms of a leaf spot, starting from the basal part of the foliage. Symptoms consisted in brown spots with not well-defined margins, irregularly circular to polygonal in shape, up to 2 to 3 mm in diameter, sometimes confluent. The internal part of spots dried. Chlorosis and yellowing surrounded the spots, involving the most part of affected leaf surfaces, due to the spread of the disease. The ornamental value of affected plants was significantly reduced. Several symptomatic leaves were washed in sterile water for 1 min. Isolations were carried out, taking several small fragments of tissues from the margins of the spots and placing them on potato dextrose agar. Fungal colonies that developed were dark green at the end of development. Successively, single-spore colonies were obtained, and pure cultures were transferred on potato carrot agar (PCA, Simmons 2007) under a light/dark regime of 11 h/13 h. On this medium, colonies formed branched conidiophores with brown, multicellular conidia, ovoid to obclavate in shape, containing one to four (average 2.4, n = 50) transverse septa and zero to one (average 0.1, n = 50) longitudinal septa. Conidia measured 10.8 to 29.3 × 5.3 to 11.2 µm (average 18.7 × 7.6 µm, n = 50). If present, the beak was 2.1 to 11.0 µm (average 5.5 µm) long. These morphological characteristics permitted identifying the fungus isolated from P. maculata as Alternaria section alternata (Woudenberg et al. 2015). The fungal DNA from the 19/33 isolate was extracted using the E.Z.N.A. Fungal DNA Mini Kit (Omega Bio-Tek, Darmstadt, Germany), and PCR was performed using the ITS (White et al. 1990), the rpb2, and the endoPG primers (Woudenberg et al. 2015). Three sequences with 529 bp (ITS), 889 bp (rpb2), and 452 bp (endoPG) (GenBank accession nos. MN871825, MN871979, and MN871978, respectively) were produced. A BLASTn analysis of these sequences exhibited 100% identity with the reference strain CBS 117.44 of A. alternata (Fries) Keissler in rpb2 and endoPG portions (accession nos. KP124772 and KP124001, respectively) and 99.6% identity in ITS region (accession no. KP124303). One P. maculata fungal isolate (19/33) was grown on PCA. A spore suspension obtained from 15-day-old cultures was sprayed at the concentration of 10⁵ CFU/ml onto leaves of three healthy plants of P. maculata. Three control plants were treated with sterile water. All plants were closed in moist plastic bags for 7 days and maintained at temperatures ranging from 15 to 28°C. The first symptoms of leaf spot appeared 10 days after the inoculation, and A. alternata was reisolated from affected leaves. Control plants remained symptomless. The pathogenicity test was repeated once using the isolate 19/40, obtaining the same results. To our knowledge, this is the first report of A. alternata on P. maculata in Italy as well as worldwide. The spread of A. alternata on P. maculata and the difficulty to manage this disease, particularly in public gardens, may discourage the use of this ornamental in Italy.

First Report of Leaf Spot Caused by a Provisionally Novel Species of Pseudocercospora on Ligustrum japonicum in South Korea

First Report of Leaf Spot Caused by a Provisionally Novel Species of <i>Pseudocercospora</i> on <i>Ligustrum japonicum</i> in South Korea

First Report of Leaf Spot on Scaevola taccada Caused by Alternaria longipes in China

First Report of Leaf Spot on <i>Scaevola taccada</i> Caused by <i>Alternaria longipes</i> in China

First Report of Leaf Blight on Aucuba japonica Caused by Nothophoma quercina in China

First Report of Leaf Blight on <i>Aucuba japonica</i> Caused by <i>Nothophoma quercina</i> in China

First Report of Leaf Spot Caused by Alternaria tenuissima on Conyza canadensis in China

First Report of Leaf Spot Caused by <i>Alternaria tenuissima</i> on <i>Conyza canadensis</i> in China

First report of leaf spots in Caryota mitis L. caused by Alternaria alstroemeriae in Pakistan

First report of leaf spots in Caryota mitis L. caused by Alternaria alstroemeriae in Pakistan

Identification of Alternaria alternata as the Leaf Spot-Causing Agent of Chicory in China

Chicory (Cichorium intybus L.) is a perennial Asteraceae herb that has been widely cultivated as a forage for livestock in many provinces of China. Since 2009, leaf spots have been observed on chicory in Chongqing, China. In April 2018, severe leaf spotting was observed at an incidence of ∼75% in a chicory (cv. Jiangjun) field in Rongchang district, Chongqing. Infected leaves initially exhibited small brown spots and then enlarged into larger, circular to elliptic, brown necrotic lesions of 5 to 15 mm diameter that coalesced to form large necrotic areas. Forty symptomatic leaves were collected and surface sterilized with 0.1% HgCl₂ solution for 3 min, rinsed three times with sterile distilled water, and air dried. Small (3 to 5 mm) sections of symptomatic leaf tissue that included the leading edge of the lesion were placed on potato dextrose agar. A fungus was consistently isolated from symptomatic leaf samples (87.5% isolation rate). Colonies were initially white and then turned gray to grayish black. Conidia were pale to brown, obpyriform to long ellipsoid, slightly constricted near some septa, with a body length of 10 to 26 µm and a body width of 6 to 14 µm. Conidia had one to four transepta and zero to three longisepta. Conidiophores were straight or flexuous, light brown to brown, and measured 10 to 28 × 2 to 5 µm. Morphological characteristics matched the descriptions of Alternaria alternata (Woudenberg et al. 2013). To confirm the identity of the fungus, the internal transcribed spacer region (ITS), the 28S large subunit ribosomal RNA (LSU), actin (ACT), and major allergen Alt a 1 (Alt a 1) genes were amplified with primers ITS1/4, LSU1Fd/LR5, ACT-512F/783R, and Alt-for/rev, respectively, of eight representative isolates were sequenced (Woudenberg et al. 2015). BLAST results showed 100% identity of the ITS (526/526 nucleotides), LSU (900/900 nucleotides), and ACT (219/219 nucleotides) sequences with those of A. alternata (KX064978, MH877754, and MH892480) and 99.78% identity of the Alt a 1 (463/464 nucleotides) sequence with A. alternata (MG598797). Based on morphology and DNA sequence analysis, the associated fungus was A. alternata. Representative sequences of one isolate (ACH2) were uploaded to GenBank (MK893994, MK896854, MK896856, and MK896855). Pathogenicity tests were performed on 10 heathy, 1-month-old potted chicory plants by spraying leaves with 5 ml of a conidial suspension (approximately 10⁵ conidia/ml) of isolate ACH2. Control plants were inoculated with sterile distilled water. After 7 days, similar leaf spots to those in the field were observed on inoculated leaves. No leaf spots developed on control plants. A. alternata was reisolated from inoculated leaves, thus completing Koch’s postulates. A. alternata was previously reported on chicory in Greece, Italy, and Turkmenistan (Farr and Rossman 2019). However, to our knowledge, this is the first report of leaf spot on chicory caused by A. alternata in China. Chicory is an important forage in south China. This disease has affected forage quality, reduced yield, and resulted in economic losses to planting enterprises and farmers. Therefore, effective prevention and control measures are urgently needed.

Pseudocercospora exilis Causing Leaf Spot Disease on Brassica rapa subsp. parachinensis in China

Chinese flowering cabbage (Brassica rapa L. subsp. parachinensis) is an important vegetable crop of China and widely cultivated in the southern provinces. During February of 2017 to 2019, a severe leaf spot disease was seen on Chinese flowering cabbage plants in different agricultural fields around Lianzhou and Guangzhou cities of Guangdong province, China. Disease incidence reached up to 70% in severely infested fields. Early disease symptoms observed were small circular or irregular spots of brown color, 3 to 11 mm in diameter and often coalesced. Ten symptomatic leaves were cut into small pieces (5 × 5 mm), surface sterilized with 1% NaOCl, plated onto potato dextrose agar (PDA) medium, and incubated at room temperature for 7 days under continuous fluorescent light. A total of 11 pathogen isolates were purified by successively subculturing on new PDA medium plates using the single-spore technique. A cercosporoid fungus was consistently isolated from diseased leaves. The colonies were raised and greenish black in color. Conidiophores were brown, subcylindrical, multiseptate, straight or slightly curved, unbranched, and smooth. Conidiogenous cells were pale brown, smooth, and arranged terminally. Conidia were pale brown, smooth, obclavate or fusoid, straight to slightly curved, with obtuse apex, one- to seven-septate, and 44.3 to 69.12 × 4.8 to 6.2 μm (n = 20). The morphological and cultural characteristics of all isolates were consistent with Pseudocercospora exilis Hern.-Gut. & Dianese (Hernandez-Gutierrez and Dianese 2009; Silva et al. 2016). One monosporic isolate (PE-1) was obtained by plating individual spores on V8 agar medium. To further confirm the species, genomic DNA was extracted from the hyphal mass of the isolate PE-1 using the DZup fungal DNA extraction kit (Sangon Biotech, Shanghai, China). The internal transcribed spacer (ITS) and translational elongation factor (EF-1α) regions of the isolate PE-1 were amplified with primers ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999) and sequenced by Sangon Biotech. The obtained DNA sequences were deposited in GenBank (accessions MN971630 for the ITS sequence and MN991198 for the EF-1α). BLAST analysis showed that the ITS and EF-1α sequences shared 99% nucleotide identity with the previously submitted P. exilis sequences (GenBank accessions NR147302 and KT290193 for ITS and EF-1α, respectively). Pathogenicity testing was performed by spraying conidial suspension (1 × 10⁶ conidia/ml) of isolate PE-1 on the leaves of 40-day-old Chinese flowering cabbage plants. Five plants were used for inoculation. Another five plants were sprayed with distilled sterilized water to serve as a control. Plants were kept in transparent boxes for 2 days to retain moisture and subsequently incubated in a growth room at 28 ± 2°C with a 12-h photoperiod. Similar leaf spots were observed on the leaves of all inoculated plants after 1 week of inoculation. No symptoms were seen on control plants. The same pathogen was reisolated consistently from inoculated leaves. The species identity was confirmed by both morphological and molecular characteristics, thus fulfilling Koch’s postulates. Previously, P. exilis has been reported to cause leaf spot disease on Chamaecrista orbiculata in Brazil (Hernandez-Gutierrez and Dianese 2009). To our knowledge, this is the first report of leaf spot on Chinese flowering cabbage caused by P. exilis. This disease can cause severe economic losses, because Chinese flowering cabbage is mainly sold in the market and used in salads or cooked dishes. Timely measures must be taken to manage this disease.

Occurrence of Leaf Spot Associated with Alternaria alternata on White Clover in China

White clover (Trifolium repens L.) is a perennial legume widely cultivated as forage, lawn grass, a green manure crop, and soil conservation plant worldwide (Zhang et al. 2016). In March 2018, necrotic leaf lesions were observed in several white clover fields in Chongqing, southwest China. The disease incidence varied from 10 to 65% between fields. Infected leaves had small, brown, circular spots that became elliptic to irregular. The spots were 1 to 15 mm in diameter with dark brown margins, yellow halos, and sometimes concentric rings. As the disease progressed, lesions gradually coalesced, forming large necrotic patches. In advanced infections, severe disease led to defoliation. Symptomatic leaves were collected from eight different fields in the Rongchang and Qianjiang districts of Chongqing, surface sterilized with 70% ethanol for 30 s followed by 0.1% HgCl₂ treatment for 3 min, and rinsed in sterile water three times. Thereafter, tissue samples (2 × 2 mm) from margins of individual lesions were placed on potato dextrose agar (PDA) amended with 50 mg/liter of chloramphenicol and incubated at 25°C in the dark. Fungal colonies consistently isolated on PDA were initially white and then turned grayish black (93.75% isolation frequency). Conidia were light to dark brown, ovoid to long ellipsoid, with one to four transverse and zero to two longitudinal septa, slightly constricted near some septa, and approximately 12 to 29 × 6 to 17 µm in size. Conidiophores were straight or flexuous, brown, and 11 to 60 × 3 to 8 µm. Morphological characteristics matched the descriptions of Alternaria alternata (Woudenberg et al. 2013). To confirm the identity, the internal transcribed spacer region (ITS), partial sequencing of 28S large subunit ribosomal RNA (LSU), major allergen Alt a 1 (Alt), and glyceraldehyde-3-phosphate dehydrogenase (GPD) genes of eight representative isolates were amplified with primers ITS1/4, LSU1Fd/LR5, Alt-for/rev, and gpd1/2, respectively, and were sequenced (Woudenberg et al. 2015). BLAST results showed 100% identity of the ITS, LSU, and Alt sequences and 99.83% identity of the GPD sequences with those of A. alternata strain CBS 104.26 (KP124299, KP124450, KP123848, and KP124156). Based on morphology and DNA sequence analysis, the associated fungus was identified as A. alternata. Representative sequences of one isolate (ATH1-2) were deposited in GenBank (MK894882, MK903026, MK903027, and MK903028). Pathogenicity tests were carried out on potted white clover, cultivar Gede. Ten healthy plants were sprayed with a conidial suspension (approximately 10⁵ conidia/ml), and the control plants were inoculated with sterile distilled water. Plants were incubated in a greenhouse at 20 to 24°C under natural light and enclosed in plastic bags for the first 2 days to maintain high humidity. After 7 days, characteristic dark brown spots were observed on the inoculated leaves and not on the control plants. A. alternata was reisolated from inoculated symptomatic leaves, thus completing Koch’s postulates. To our knowledge, this is the first report of leaf spot on white clover caused by A. alternata in China. This disease not only seriously affects lawn appearance and quality but also greatly reduces forage quality and yield, resulting in economic losses to animal husbandry.

First report of leaf spot on Amorphophallus muelleri caused by Colletotrichum gloeosporioides in Yunnan, China

First report of leaf spot on Amorphophallus muelleri caused by Colletotrichum gloeosporioides in Yunnan, China

First Report of Leaf Spot of Chenopodium album Caused by Fusarium equiseti in China

Chenopodium album L. (commonly known as bathu) is one of the most troublesome weeds associated with crops in the world. In June 2018, leaf spot on C. album was observed in soybean and corn fields in Yanshou County (45°26′N, 128°18′E), Heilongjiang Province, China, with 20 to 40% incidence. The disease was not observed on soybean or maize. Initial symptoms were brown necrotic spots with a white center. Lesions rapidly expanded (1 to 10 mm diameter), contained concentric rings, and became necrotic. Ten C. album leaves were collected for fungal isolations. Diseased tissues were surface sterilized with 0.1% HgCl₂ for 1 min, rinsed in sterile distilled water, and cultured on potato dextrose agar at 26°C. After 5 days, six fungal isolates were obtained and subcultured by transferring hyphal tips. The colony had abundant white aerial mycelia and became peach-orange in color with age on potato dextrose agar. Macroconidia were mostly five- to seven-septate, slightly curved with a tapered and elongated apical cell and prominent foot-shaped basal cell, 23.2 to 31.8 μm in length, and 3.1 to 4.92 μm in width on carnation leaf agar medium. No microconidia were observed. Chlamydospores were abundant in clumps or chains, ellipsoidal or subglobose. Morphological characteristics of the isolates were consistent with those of Fusarium equiseti (Jacobs et al. 2018). Genomic DNA was extracted from single conidial cultures of the representative isolate H5, and the internal transcribed spacer regions (ITS) and translation elongation factor 1-α gene (TEF1) were amplified with primers ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Akbar et al. 2018), respectively, and sequenced. The DNA sequence of the isolate was identical, and the representative isolate H5 was deposited in GenBank (accession nos. MN241064 and MK990601). BLAST analysis showed that the obtained sequence for the ITS amplicon shared 99.6% similarity with F. equiseti isolate KA (accession no. JQ690085.1), whereas the sequence of the TEF1 amplicon shared 99% similarity with F. equiseti strain G17WX1-5-2 (accession no. MG670196.1). To determine pathogenicity, C. album plants were grown in 15-cm pots containing a commercial potting mix (one plant/pot). After the eight-leaf stage, three plants (two leaves/plant) were inoculated with 50 μl of a conidial suspension (4 × 10⁶ spores/ml). Three plants treated with sterile distilled water served as a control. All plants were placed in a humidity chamber (>95% relative humidity) at 26°C for 48 h after inoculation and then kept in a greenhouse at 22/28°C (night/day). After 10 days, all inoculated leaves showed symptoms similar to those observed in the field. No disease occurred on control plants. Five same fungal isolates were reisolated from five diseased leaves and confirmed to be F. equiseti. To our knowledge, this is the first report of leaf spot on C. album caused by F. equiseti in China.

Identification and Characterization of Gonatobotryum apiculatum Causing Leaf Spot and Blight on Sinowilsonia henryi

Sinowilsonia henryi is a rare and endangered plant, as well as an endemic species in China. In July 2018, leaf spot and blight disease was observed on S. henryi in Yichang, Hubei, China. A fungus isolated from disease tissues was identified as Gonatobotryum apiculatum based on morphology and sequence analyses of ITS and LSU regions. Phylogenetic analyses indicated that the species belongs to Dothioraceae (Dothideales). Morphologically, the species produced two distinct types of conidia from authentic media, both conidia were described here. Pathogenicity tests showed that the fungus is a pathogen causing leaf spots on S. henryi. This is the first report of leaf spot and blight disease on S. henryi caused by G. apiculatum in China.