Partial Β-tubulin Sequences Research Articles

Re-identification of Strains from Aspergillus Section Aspergillus and Description of Three Unrecorded Species from Korea

The section Aspergillus includes xerophilic fungi that are economically significant and broadly distributed in natural settings as well as human habitats and are recognized for their sustenance on substrates with low water activity. Accurate identification of fungal species is essential for any reliable advances in mycological research. In this study, 108 strains from the section Aspergillus, originating from Korea and conserved at the Korean Agricultural Culture Collection, were subjected to re-identification using a combined dataset that included partial sequences of β-tubulin (BenA), Calmodulin (CaM), and RNA polymerase II second largest subunit (RPB2) genes, along with their morphological characteristics. We confirmed the presence of 12 species among the 108 strains originally isolated from Korea. Of them, nine species have been formerly reported in Korea (Aspergillus chevalieri, Aspergillus cibarius, Aspergillus cumulatus, Aspergillus glaucus, Aspergillus montevidensis, Aspergillus proliferans, Aspergillus pseudoglaucus, Aspergillus ruber, and Aspergillus tonophilus), and 3 species (Aspergillus aurantiacoflavus, Aspergillus intermedius, and Aspergillus niveoglaucus) were found to be previously unreported to be isolated from Korea. Here, the detailed characteristic features of these three unexplored species are presented, including specific morphological traits, genetic variations, and ecological niches in Korea.

First Report of Colletotrichum gloeosporioides Causing Anthracnose on Peanut in Chongqing, China.

In July 2022, dieback and discoloration were detected on infected stems of peanut in Qijiang District of Chongqing (106.56°E,29.41°N), China, with an incidence up to 5%. These peanut stems had disease symptoms typical of anthracnose with irregular gray-brown spots with dark brown edges, sunken, and necrotic. High temperature and high humidity were favorable for the growth of the pathogen. To isolate the pathogen, we collected 10 typical infected peanuts and cut one piece from each of symptomatic stems, surface sterilized with 0.5% NaClO for 1 min, and 75% ethanol for 30 s, then rinsed three times with sterile distilled water and dried on sterilized filter paper. These pieces were incubated on potato dextrose agar (PDA) at 25°C in the dark. Pure cultures were obtained from hyphal tips of each colony. It was found that isolates with the same colony morphology were isolated from each infected stem. A representative isolate (L7) was used for morphological characterization, molecular analysis, phylogenetic analysis, and pathogenicity tests. The colonies appeared white to gray, with white margins and aerial hyphae, and the reverse of the colonies was gray to brown. Conidia were cylindrical, aseptate, with obtuse to slightly rounded ends, 13.4 to 18.8 × 4.2 to 5.8 μm (n=50). Morphological characteristics were generally consistent with those of Colletotrichum gloeosporioides species complex (Cannon et al., 2012). For molecular identification, genomic DNA was extracted using a CTAB method and partial sequences of β-tubulin (TUB2), actin (ACT) genes, chitin synthase (CHS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were amplified and sequenced using primers T1/T2, ACT-512F/ACT-783R, CHS-79F/CHS-345R, and GDF1/GDR1, respectively (Damm et al., 2012; Dowling et al., 2020). Using the BLAST, TUB2, ACT, CHS and GAPDH gene sequences (GenBank accession No. OR714793, OP168707, OP168708 and OR714794, respectively) were100% (429 bp out of 429 bp), 99.22% (256 bp out of 258 bp), 99.64% (276 bp out of 277 bp) and 100% (253 bp out of 253 bp) identical to C. gloeosporioides CBS:112999 (JQ005587, JQ005500, JQ005326, and JQ005239), respectively. Using Neighbor-Joining algorithm, phylogenetic analysis was conducted based on the concatenated sequences of published TUB2, ACT, CHS and GAPDH genes. The identified isolate (L7) was closely related to C. gloeosporioides. To evaluate pathogenicity, the stems of ten peanut (Zhonghua12) seedlings (2 weeks) were wounded with a sterile toothpick and mycelial plugs (5 mm in diameter) or 20 μl of conidial suspension (105/ml) were inoculated. Non-colonized agar plugs or 20 μl of sterile distilled water were treated as control. After inoculation, the peanuts were kept in a moist chamber at 28°C with 80% humidity in the dark for 24 h, and subsequently transferred to the moist chamber with 12 h light and darkness cycle for 6 days, similar symptoms were observed on all inoculated peanuts. Controls remained asymptomatic. C. gloeosporioides was reisolated from the diseased stems and confirmed using morphological features and sequence analysis of TUB2, ACT, CHS and GAPDH. Anthracnose caused by C. truncatum and C. fructicola has been reported on peanut leaves in China (Gong et al., 2023; Yu et al., 2019). To our knowledge, this is the first report of anthracnose on peanut stem caused by C. gloeosporioides in Chongqing. Our report will provide crucial information for studying on epidemiology and management of this disease.

Branch blight of Juglans sigillata caused by Juglanconis appendiculata in China.

Juglans sigillata Dode, an endemic walnut species native in southwest China, is mainly used as nuts in Sichuan Province (Jin et al., 2019). In May 2021, symptoms of branches blight were observed in an orchard measuring 10 hectares located in Mianyang City, Sichuan Province (31°5' 25″N, 105°27'36″E, 365 m above sea level). About 40% of plants were diseased in the quadrat consisting of twenty walnut trees, and 20% of branches were dead on each affected tree. Initially, light brown spots appeared; then, the spots expanded to surround the whole branches; finally, the branches changed from brown to reddish-brown and died. Four symptomatic branches were sampled randomly from different trees. Next, four fungal isolates were obtained from the acervuli of each branch using the single-conidium isolation (Chomnunti et al. 2014) and cultured on potato dextrose agar (PDA). The Petri dishes were placed in an incubator and cultured at 25 °C under a 12-h photoperiod. Colonies were initially white with thin aerial mycelia and gradually turned dark grey with irregular margins. Conidiomata were acervular, black and scattered, with a diameter of 0.3 - 0.7 mm. Conidiophores were narrowly cylindrical, simple or branched at the base, 30 - 43 × 3 - 8 µm (x = 36.5 × 5.5 µm, n = 40). Conidiogenous cells were annellidic with distinct annellations. Conidia were unicellular, brown when mature, narrowly ellipsoid with gelatinous sheaths and truncate scars at the base, 17 - 32 × 7 - 12 µm (x = 27 × 9 µm, n = 40). The genomic DNA of a representative isolate SICAUCC 22-0064 was extracted, and the internal transcribed spacer (ITS) region, guanine nucleotide-binding protein subunit beta gene (ms204), translation elongation factor 1-alpha (tef1-α), and partial sequences of β-tubulin (tub2) were amplified by polymerase chain reaction and sequenced with primers V9G/LR5 (de Hoog & van den Ende 1998), MS-E1F1/MS-E5R1 (Walker et al. 2012), EF1-728F (Carbone & Kohn 1999)/TEF1LLErev (Jaklitsch et al. 2005), and T1/BtHV2r (Voglmayr et al. 2017), respectively. The sequences of ITS, ms204, tef1-α, and tub2 were deposited in NCBI with accession numbers ON000068, ON112376, ON112374, and ON112375, respectively. With the consideration of the sequence lack of ms204 and tub2 in the ex-type strain (D96) of Juglanconis appendiculata Voglmayr & Jaklitsch, the isolate D140 was used for nucleotide blast. The results showed 99.68%, 100%, 100%, and 100% identities of ITS, ms204, tef1-α, and tub2 with D140 (accession numbers KY427138, KY427157, KY427207, KY427226). Phylogenetic analysis based on a combined dataset showed 100% bootstrap with J. appendiculata, and the morphology was consistent with the asexual stage of J. appendiculata (Voglmayr et al., 2017). To verify Koch's postulates, five branches wounded by pin-prick were sprayed with conidial suspension (1 × 105 conidia/mL) in each plant, and three repetitions were performed on healthy 2-year-old potted plants. The same number of branches were sprayed with sterile distilled water as controls. The plants were placed in a greenhouse at 25 ℃ under 90% relative humidity and a 12-h fluorescent light/dark regime. After five weeks, all the inoculated branches showed brown necrosis similar to that observed in the field, and no symptoms occurred on the controls. The pathogens were re-isolated from the necrotic lesions and identified by morphology and phylogeny. J. appendiculata has been reported on Juglans nigra and J. regia in Austria, France, Spain and Greece (Farr & Rossman 2022). This paper is the first report of branch blight on Juglans sigillata caused by J. appendiculata in China. This result may develop the understanding of walnut diseases and lay a foundation for further management.

Leaf Blight on Photinia × fraseri Caused by Pestalotiopsis trachicarpicola in China.

Photinia × fraseri is a well-known green plant mainly distributed in the Yangtze River and Yellow River Basin, east and southwest of China (Guan et al. 2013). In October 2020, typical leaf blight symptoms on roughly 10% leaves in a Photinia × fraseri shrub were observed in the campus of Sichuan Agricultural University (30°42'19″ N, 103°51'29″ E). Initially, chlorotic lesions with brown margins occurred on the leaf margin, then the large patches formed to cause leaves necrotic, finally lesions to dry and acervulus bred in 2-4 months later. Five single conidium isolates were carried out (Chomnunti et al. 2014) cultured on potato dextrose agar (PDA) at 25 ℃. All isolates shared similarly morphological characteristics, which was white and thin, and the reverse were yellowish. Mycelium was hyaline, sparsely septate, measuring 1-4 μm in diam. Conidiogenesis formed after 7 days. Conidiogenous cells were discrete, lageniform, smooth, thin-walled, colorless. Conidia were fusiform, straight to slightly curved, 4-septate, 21-30 × 5-7 μm (x ̅= 27 × 6.0 μm, n=30); basal cells were obconic with truncate base, hyaline, thin- and smooth-walled, 4-7 μm long (x ̅= 5.5 μm, n=30); three median cells were doliiform with thick walls, concolorous, olivaceous, constricted at the septa, and septa and periclinal walls were darker than the rest of the cell, 14-20 μm long (x ̅= 17 μm, n=30); apical cells were hyaline, conic to cylindrical, 3.0-6.5 μm long (x ̅= 4.5 μm, n=30), with 2-4 (mostly 3) tubular apical appendages arising from the upper portion, rarely branched, 7.5-18 μm long (x ̅= 12 μm, n=50); basal appendage was single, unbranched, 3-10 μm long (x ̅= 6.5 μm, n=30). DNA was extracted from the representative strain (SICAUCC 21-0012), and the internal transcribed spacer (ITS) region, the large subunit of the nrDNA (LSU), translation elongation factor 1-alpha (tef1-α), and partial sequences of β-tubulin (tub2) were amplified by polymerase chain reaction and sequenced with primers ITS5/ITS4, LR0R/LR5, 728F/1567R, and Bt2a/Bt2b, respectively (Zhang et al. 2012, Ariyawansa & Hyde 2018). The sequences were deposited in GenBank, viz. MZ453106, MZ453108, MZ467300, MZ467301, respectively. The nucleotide blast showed 99% (ITS, 0 gaps), 100% (tub2, 0 gaps), 100% (tef1-α, 0 gaps) identities with the ex-type Pestalotiopsis trachicarpicola Yan M. Zhang & K. D. Hyde (IFRDCC 2440). The fungus was identified as P. trachicarpicola combined with phylogeny and morphology (Maharachchikumbura et al. 2012, Zhang et al. 2012). To conduct Koch's postulates, five healthy 6-year-old P. × fraseri were inoculated with 10 µl spore suspension (106 conidia/ml) onto the wounded sites (five leaves per plant, ~1 to 2 years old) via sterile pin, and five healthy plants treated with sterile dH2O as controls (Yang et al. 2021). The plants were placed in a greenhouse at 25°C with relative humidity >80%. After 2 months, leaf blight symptoms gradually emerged on inoculated leaves, and the controls were symptomless. Fungal isolates from symptomatic plants showed similar morphological characteristics as SICAUCC 21-0012, and the pathogen was not isolated from asymptomatic plants. To our knowledge, this is the first report of leaf blight caused by P. trachicarpicola on Photinia × fraseri in China. Disease management should be adopted properly to restore and improve its ornamental value.

Colletotrichum aenigma Associated with Apple Bitter Rot on Newly Bred cv. RubyS Apple

The abnormal brown sunken lesions were observed on cv. RubyS apple fruits in an orchard located in Gunwi, Gyeongbuk province, Korea. The primary observed symptoms such as small round sunken lesions and small black dots on the symptomatic area were different from the reported apple diseases. The affected apple fruits were sampled and subjected to isolation of the causal agent. Cultural and morphological characteristics of isolated fungal strain, designated KNUF-20GWA4, were similar to that of Colletotrichum spp. Based on multilocus sequence analyses using internal transcribed spacer regions and partial sequences of β-tubulin, glyceraldehyde-3-phosphate dehydrogenase, chitin synthase, and actin genes, strain KNUF-20GWA4 showed 99.2-100% similarities with C. aenigma ICMP 18608 and the isolate clustered together with several other strains of this species in the phylogenetic tree. To our knowledge, this is the first report of bitter rot on apple fruits caused by C. aenigma.

First Report of the Colletotrichum siamense species complex causing Anthracnose on Photinia × fraseri Dress in China.

Photinia × fraseri Dress is mainly distributed in the southeast and east of Asia and North America and has been widely cultivated in China. In summer 2018, an anthracnose disease of P. × fraseri Dress was found in a park in Nanjing City, China. Disease leaves showed small, round, light reddish brown spots in the early stage of infection that gradually expanded into round spots, with light gray in centers and brown edges. Fresh lesions were cut into 2-3 mm2 sections, sterilized with 75% ethanol for 30 s, followed by 1% NaClO for 90 s, washed with sterile water 3 times, and placed on potato dextrose agar (PDA) with 0.1 mg/mL ampicillin at 25°C. Colonies of a representative strain "HDSN2-1" were white to greenish grey, and the daily growth rate was 9.5 to10.5 mm/day. Aerial mycelium was grayish white, dense, and cottony, with visible conidial masses at the inoculum point. Conidia were one-celled, smooth-walled, hyaline, with obtuse to rounded ends, with a size of 12.8 to 18.4 × 4.5 to 6.8 µm. Appressoria were one-celled, brown, thick-walled, ellipsoidal, and 7.3 to 10.3 × 5.4 to 6.97µm. The morphological characteristics of HDSN2-1 matched those of the Colletotrichum gloeosporioides species complex (Weir et al. 2012). For further identification, DNA was extracted from HDSN2-1 mycelium and the internal transcribed spacer (ITS) region and partial sequences of β-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-1) and calmodulin genes(CAL) were amplified by PCR, and sequenced with primers ITS1/ITS4, TubF1/TubR1, GDF/GDR, CHS-79F/CHS-345R, and CAL1C/CAL2C, respectively(Weir et al. 2012). The sequences were deposited in GenBank [Accession nos: MN889417, MN894596, MN894597, MN894598, MN894599]. A phylogenetic tree was constructed using the maximum likelihood/span>method with concatenated sequences (ITS, TUB2, GAPDH, CHS and CAL) (Zhu et al. 2019). Analyses conducted in MEGA7 placed HDSN2-1 in the C. siamense clade, which includes ex-type ICMP 18578. Pathogenicity of HDSN2-1 was verified on leaves from 7 healthy 8-year-old P. × fraseri and inoculated with either 5-mm mycelial plugs from the edge of 5-day old cultures on PDA or 10 μL of spore suspension (106 conidia/mL),15 healthy plants（8-year-old）were used in 5 repetitions (5 for control, and 10 for the pathogenicity test) in the same way. Controls were treated with PDA plugs or sterile dH2O. The leaves were incubated at 25 ℃ and the inoculated plants were kept in a greenhouse (relative humidity > 85%, 25 ± 1°C). Symptoms were not observed on control plants. Fungal isolates from the symptomatic plants showed the same morphological characteristics with HDSN2-1. C. siamense is a common fungal pathogen of many plants. For example, it was previously reported infecting apples and citrus fruits ( Abirammi et al. 2019). This is the first report of anthracnose of P. × fraseri caused by C. siamense in China.

New Endophytic Fusarium spp. from Fraxinus excelsior Leaves in Slovakia.

Fraxinus excelsior is currently suffering from ash dieback disease caused by the fungus Hymenoscyphus fraxineus. Co-occurrence of large numbers of other fungi with endophytic as well as pathogenic lifestyles has been observed on F. excelsior. In this study, new endophytic Fusarium spp. isolate Fraxinus excelsior Fusarium from Slovak territory, associated with F.excelsior, was characterised. Using morphology-based approaches, the fungus was assigned to the genus Fusarium. The isolate formed thick-walled hyaline, fusiform, slightly curved macroconidia, produced from monophialides in sporodochia, pointed at the tip, mostly 3 - 5 septate, occasionally 6 septate, and 40 - 68 × 3.8 - 5.0 µm in size with basal pedicellate cells. For more accurate taxonomic affiliation, molecular sequence analyses of the internal transcribed spacer, translation elongation factor 1-alpha sequences, and partial β-tubulin gene were performed. While the internal transcribed spacer sequence of the isolate showed the highest similarity to the Fraxinus oxysporum species, translation elongation factor 1-alpha and partial β-tubulin sequences were distantly related to the Fraxinus avenaceum species. Based on these data, the Fraxinus excelsior Fusarium isolate could probably be considered a new species of the Fusarium genus.

Five Fungal Pathogens Are Responsible for Bayberry Twig Blight and Fungicides Were Screened for Disease Control.

Bayberry (Myrica rubra) is a commercial fruit in China. For the past seven years, twig blight disease has been attacking bayberry plantations in Shantou City, Guangdong Province, China, leading to destructive damage and financial loss. In this study, five fungal species associated with twig dieback and stem blight were identified based on morphological characteristics combined with multilocus sequence analysis (MLSA) on the internal transcribed spacer (ITS) region, partial sequences of β-tubulin (tub2), translation elongation factor 1-α (tef1-α), large subunit ribosomal RNA (LSU) and small subunit ribosomal RNA (SSU) genes, which are Epicoccum sorghinum, Neofusicoccum parvum, Lasiodiplodia theobromae, Nigrospora oryzae and a Pestalotiopsis new species P. myricae. P. myricae is the chief pathogen in fields, based on its high isolation rate and fast disease progression after inoculation. To our knowledge, this is the first study reporting the above five fungi as the pathogens responsible for bayberry twig blight. Indoor screening of fungicides indicates that Prochloraz (copper salt) is the most promising fungicide for field application, followed by Pyraclostrobin, 15% Difenoconazole + 15% Propiconazole, Difenoconazole and Myclobutanil. Additionally, Bacillus velezensis strain 3–10 and zeamines from Dickeya zeae strain EC1 could be used as potential ecofriendly alternatives to control the disease.

First Report of Sclerotinia Blight Caused by Sclerotinia sclerotiorum on Oriental Tobacco in the Yunnan Province of China

Yunnan Province is a major oriental tobacco (OT) production area with >90% of China’s output (Qu et al. 2014). In the spring of 2018 and 2019, we conducted OT disease surveys in Mengtong, Baoshan, Yunnan Province, and we found an unknown disease displaying rotting, wilting, and blighting in two fields (24°32′37″ N, 99°37′34″ E; 24°32′46″ N, 99°37′31″ E; ∼2,000 m²). Average incidence was 3.0 and 8.4%, respectively. Disease development began with water-soaked, oval or circular spots with white centers and olive-green edges that formed on the wounds of stems and petioles near the soil, and infected stems subsequently became bleached and died. In the late phases, white cottony mycelium appeared on the dead tissues. Spherical to cylindrical black sclerotia (5.0 to 17.0 × 3 to 7 mm; avg. 9.1 × 4.5 mm; n = 50) were observed inside and on the surface of stems. To isolate the pathogen, five and four infected samples were collected from fields in 2018 and 2019, respectively. Tissue sections (5 mm²) with typical symptoms were surface disinfected in 75% ethanol for 5 s and 0.5% NaOCl for 1.5 min, rinsed in sterilized distilled water three times, and dried. Tissues were placed on PDA plates and cultured at 25°C for 3 days in the dark. Hyphal tips were transferred to fresh PDA plates. Thin, cottony-white, fluffy mycelia grew to cover the Petri dish (9 cm diam.) in 3 to 4 days. Irregular white immature sclerotia were produced 4 to 5 days later on the edges of the plates. After 7 to 14 days, abundant spherical or oval black sclerotia of 2.4 to 5.0 × 1.4 to 3.8 mm (avg. 3.4 × 2.6 mm, n = 50) were formed. Morphology of all isolates was similar to Sclerotinia sclerotiorum (Lib.) de Bary. To confirm the pathogen, we extracted genomic DNA from the mycelia of isolate HP-1 using a Fungal DNA Extraction Kit (Omega). The ITS region and partial β-tubulin gene sequence of HP-1 were amplified with primers ITS1/ITS4 and Bt2a/Bt2b, respectively (Doyle and Doyle 1987; Glass and Donaldson 1995). PCR amplicons were purified and cloned into pMD19-T simple vector (Takara Biomedical) for Sanger dideoxy sequencing at Shanghai Life Technology Co. BLASTn search revealed the 462-bp ITS sequence (MK656936.1) was identical to S. sclerotiorum (MK527226.1, MK010971.1, MG931017.1, etc.), and the 472-bp partial β-tubulin sequence (MN295984.1) was too (MG931018.1, KX781302.1, KF545201.1, etc.). Koch’s postulates were fulfilled by inoculation and reisolation of HP-1 from five plants. Five mycelial plugs, 8 mm diameter, from 5-day-old culture grown on PDA were used to inoculate the lower stems of five 45-day-old susceptible plants (BASMA-1) and wrapped with wet sterilized cotton. PDA plugs without fungi were controls. Plants were maintained in a greenhouse at 25°C with a 12-h photoperiod. Five days after inoculation, all inoculated stems became water-soaked brown and covered with white mycelia. Symptoms typical of Sclerotinia blight (SB) on stems were observed 10 days after inoculation. S. sclerotiorum was consistently reisolated. No symptoms developed on the controls. Similar results were obtained in three independent experiments. Seedling blight caused by S. sclerotiorum has been recorded on flue-cured tobacco in Brazil, the United States, and Jilin and Anhui Provinces of China (Gao et al. 2018; Farr and Rossman 2019; Qian et al. 1994). This is the first observation of SB on OT in Yunnan Province. OT (Nicotiana tabacum L.) is a sun-cured, highly aromatic, small-leafed variety planted under dry and hot conditions from November to April in Baoshan, where rainfall increases from February to April. SB is a major disease of OT in this area; however, it has not been reported on flue-cured tobacco in Yunnan Province. SB of OT is often misidentified as black shank caused by Phytophthora nicotianae, the most common disease on flue-cured tobacco in Yunnan Province (Zhang and Chen 1999). This report will aid local farmers and tobacco technicians in accurate diagnosis and control of the disease with more appropriate measures.

Macrophomina Crown and Root Rot of Pistachio in California.

In this study, declining pistachio rootstocks were detected in newly planted commercial pistachio orchards in Kern County, California. Symptoms were characterized by wilted foliage combined with crown rot in the rootstock. From diseased trees, 42 isolates were obtained, and all had similar cultural and morphological characteristics of Macrophomina phaseolina. Analyses of nucleotide sequences of three gene fragments, the internal transcribed spacer region (ITS1–5.8S–ITS2), partial sequences of β-tubulin, and translation elongation factor 1-α (TEF1) confirmed this identification, and 20 representative isolates are presented in the phylogenetic study. Testing of Koch’s postulates showed that M. phaseolina, when inoculated to stems and roots of the pistachio rootstocks using mycelial plugs or a microsclerotial suspension, is indeed pathogenic to this host. The widely used clonal University of California Berkeley I (UCBI) rootstock appeared highly susceptible to M. phaseolina, suggesting that this pathogen is an emerging threat to the production of pistachio in California. This study confirmed the association of M. phaseolina with the decline of pistachio trees and represents the first description of this fungus as a crown rot-causing agent of pistachio in California.

First Report of Twig and Branch Dieback, Caused by Eutypa lata, on Loquat in the Western Cape Province of South Africa

HomePlant DiseaseVol. 104, No. 3First Report of Twig and Branch Dieback, Caused by Eutypa lata, on Loquat in the Western Cape Province of South Africa PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Twig and Branch Dieback, Caused by Eutypa lata, on Loquat in the Western Cape Province of South AfricaP. Moyo, L. Mostert, R. Dedekind, W. J. van Jaarsveld, R. Pierron, and F. HalleenP. MoyoDepartment of Plant Pathology, University of Stellenbosch, Matieland, 7602, South AfricaSearch for more papers by this author, L. Mosterthttp://orcid.org/0000-0001-9063-2447Department of Plant Pathology, University of Stellenbosch, Matieland, 7602, South AfricaSearch for more papers by this author, R. DedekindDepartment of Plant Pathology, University of Stellenbosch, Matieland, 7602, South AfricaSearch for more papers by this author, W. J. van JaarsveldDepartment of Plant Pathology, University of Stellenbosch, Matieland, 7602, South AfricaSearch for more papers by this author, R. PierronDepartment of Plant Pathology, University of Stellenbosch, Matieland, 7602, South AfricaSearch for more papers by this author, and F. Halleen†Corresponding author: F. Halleen; E-mail Address: HalleenF@arc.agric.zahttp://orcid.org/0000-0002-2348-1201Plant Protection Division, ARC Infruitec-Nietvoorbij, Stellenbosch, 7599, South AfricaSearch for more papers by this author AffiliationsAuthors and Affiliations P. Moyo1 L. Mostert1 R. Dedekind1 W. J. van Jaarsveld1 R. Pierron1 F. Halleen2 † 1Department of Plant Pathology, University of Stellenbosch, Matieland, 7602, South Africa 2Plant Protection Division, ARC Infruitec-Nietvoorbij, Stellenbosch, 7599, South Africa Published Online:5 Jan 2020https://doi.org/10.1094/PDIS-06-19-1284-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Loquat (Eriobotrya japonica Lindl., Rosaceae) is a subtropical tree grown for its edible fruit (Tziros 2013). In South Africa, loquat is grown as an ornamental tree. Twig and branch dieback symptoms as well as cankers extending from pruning wounds or natural openings were observed on trees during surveys conducted in the Western Cape Province, South Africa, between July 2013 and November 2014 (Moyo et al. 2019). Internal symptoms included black discoloration below wounds or along cankers, which appeared wedge-shaped in cross section. Wood pieces removed from the margin between healthy and diseased tissues were surface sterilized (70% ethanol for 30 s, 2.5% sodium hypochlorite for 2 min, and 70% ethanol for 30 s), plated onto potato dextrose agar plates containing chloromycetin (250 mg/liter), and incubated at 24°C and 12-h photoperiod. Two representative isolates (STEU 8519 and STEU 8520) were purified and characterized. The fungus had cottony-white mycelium that was cream in reverse. No sexual morph was observed in culture, but the cottony-white mycelium turned gray with age. Filiform conidia, measuring (17 to) 21 to 39 (to 42) × 1 to 2 μm (n = 60), were formed in pycnidia and exuded in cream-orange droplets. Conidiophores were branched. These characteristics fit those of Eutypa lata (Pers:Fr) Tul & C. Tul. (Munkvold 2001). The identity of the isolates was confirmed by DNA sequencing of the internal transcribed spacer (ITS) rDNA region (GenBank accession nos. MG978312 and MG978313) and partial β-tubulin gene (MG978308 and MG978309). Nucleotide BLAST analysis of ITS sequences showed 99% identity with E. lata strain CA38 (AY462541) and strain LATA1 (KR909218) for STEU 8519 and STEU 8520, respectively. Partial β-tubulin sequences were 100% similar to E. lata strain STEU 8522 (MG978307) and strain STEU 8239 (MF359689) for STEU 8519 and STEU 8520, respectively. Pathogenicity was tested on 2-year-old healthy loquat shoots on 5-year-old trees (n = 12). Three shoots per plant were surface sterilized at the point of inoculation by spraying with 70% ethanol and wounded using a 4-mm-diameter cork borer. Mycelial plugs harvested from the periphery of 14-day-old colonies were placed into two of these wounds. Noncolonized PDA plugs (control) were placed into the third wound. All inoculated wounds were wrapped with Parafilm. After 8 weeks, inoculated shoots were removed, sterilized as described above, and cut open longitudinally through the inoculation point. The mean length of discoloration, which extended in both directions from the inoculation point, measured 43.6 and 51.7 mm for STEU 8519 and STEU 8520, respectively. The inoculated fungus was reisolated from all inoculated shoots. Control shoots remained asymptomatic, and no fungus was isolated. E. lata has been reported on loquat in Australia (Rappaz 1987). This is the first report of E. lata causing twig and branch dieback of loquat in South Africa. This is a significant finding because loquat, which is often found in close proximity to vineyards and pome and stone fruit orchards, could potentially serve as an inoculum source of E. lata to these important crops.The author(s) declare no conflict of interest.

Fusarium avenaceum: A Toxigenic Pathogen Causing Ear Rot on Maize in Yunnan Province, China

<i>Fusarium avenaceum</i>: A Toxigenic Pathogen Causing Ear Rot on Maize in Yunnan Province, China

Morphology and multigene phylogeny of Talaromycesamyrossmaniae, a new synnematous species belonging to the section Trachyspermi from India.

A new Talaromyces species, T.amyrossmaniae, isolated from decaying fruit and litter of Terminalia bellerica, is described and illustrated. On the natural substrate, the new species produces determinate synnemata, with a well-defined, vivid orange red to orange red cylindrical stipe, and a greyish green capitulum. Conidiophores are typically biverticillate, or sometimes have subterminal branches, with acerose phialides that produce globose to subglobose, smooth to slightly roughened conidia. Multigene phylogenetic analyses based on the internal transcribed spacer region (ITS), and partial sequences of β-tubulin (BenA), calmodulin (CaM), and DNA directed RNA polymerase second large subunit (RPB2) genes, along with morphological characterization, revealed that these isolates are distinct and form a unique lineage of Talaromyces in section Trachyspermi, closely allied to T.aerius, T.albobiverticillius, T.heiheensis, T.erythromellis, and T.solicola. The new species T.amyrossmaniae is the first species in section Trachyspermi with determinate synnemata.

Botryosphaeriaceae causing branch dieback and tree death of macadamia in Australia

Incidence of branch dieback of macadamia with characteristic symptoms including dark necrotic lesions on the wood and blackening of the vascular tissue is increasing in commercial macadamia orchards in the south-eastern production regions in Australia. In many cases, disease progresses from the branch to the main trunk resulting in total tree death and reduced orchard productivity. Previously, only Botryosphaeria ribis was associated with branch dieback of macadamia, however, recent observations suggest other species in the Botryosphaeriaceae may be involved. This study aimed to identify the causal agents of branch dieback of macadamia and examine their pathogenicity on a main macadamia cultivar (HAES 246). Thirty-four representative Botryosphaeriaceae isolates, obtained from over 150 samples of branch dieback symptoms on macadamia trees, were identified using the partial sequence of the internal transcribed spacer of the rDNA and partial sequences of β-tubulin and elongation factor gene regions. Six species in the Botryosphaeriaceae were identified. Lasiodiplodia pseudotheobromae (n = 18; 53%) was the most prevalent species, followed by Neofusiccocum parvum (n = 5; 14%), L. iraniensis (n = 4; 12%), N. luteum and L. theobromae (n = 3; 9% each) and N. australe (n = 1; 3%). Using an in planta assay, pathogenicity tests showed that all six species caused dieback and necrotic lesion symptoms on macadamia plants. Inoculated plants died within 4 weeks of inoculation, showing the characteristic symptoms (blackening of the wood tissue). Inoculation trials revealed differences in aggressiveness among the six species.

Genetic Variability of Phyllosticta ampelicida, the Agent of Black Rot Disease of Grapevine.

Phyllosticta ampelicida causes black rot disease of Vitis spp. Genetic homogeneity of pathogen populations was investigated by analyzing the number of haplotypes present in infected samples from Europe and America. The fungus was identified from an analysis of the internal transcribed spacer (ITS)1-ITS2 region, and partial sequences of β-tubulin and calmodulin genes. The analysis of nuclear microsatellites applied to strains from Vitis spp. confirmed the existence of a high degree of genetic variability in the fungal populations, revealed four subpopulations, and showed that strains from America are distinct from the European ones. Furthermore, the results obtained by landscape genetics showed that there were different introductions of the pathogen in the main vine areas of Europe, confirming what was observed in the first reports of the disease. The genetic variability of the fungus revealed by this study confirms the ability to generate new haplotypes by sexual reproduction. The difference found between the European populations and the American one confirms that the pathogen originated from America.

Diversity of Penicillium species isolated from heavy metal polluted soil in Guizhou Province, China

Seven Penicillium strains were isolated from soil samples polluted by heavy metals in different zones of Guizhou Province, China. Phenotypic identification proved to be difficult and this data was therefore supplemented with ribosomal internal transcribed spacer region and partial β-tubulin sequences. Comparison of the obtained sequences with references sequences showed that six strain belonged to Penicillium section Lanata-divaricata and one strain to section Sclerotiora. Three of the seven isolates belonged to a new species and were named here P. terrarumae sp. nov. The other four isolates were identified as P. cremeogriseum, P. guanacastense, P. griseopurpureum and P. oxalicum.

First report of Alternaria alternata causing black spot on pink lapacho (Handroanthus impetiginosus)

A severe leaf spot disease was observed on pink lapacho trees, Handroanthus impetiginosus for the first time in Buenos Aires province, Argentina during the autumn of 2013. The pathogen was identified as Alternaria alternata based on the morphological characteristics and sequence data from the internal transcribed spacer region of ribosomal DNA, and partial β-tubulin sequence. A pathogenicity test was performed and Koch’s postulates were confirmed by re-isolating the fungus from artificially inoculated leaves. This is the first report of Alternaria black spot of Handroanthus impetiginosus trees.

First Report of Rhexocercosporidium panacis Causing Rusty Root of Panax ginseng in Northeastern China.

In northeastern China, Asian ginseng (Panax ginseng) roots exhibited reddish brown lesions of various sizes, irregular shapes, and diffuse margins, typical of rusty root disease. The lesions remain superficial, smooth, and limited to the epidermal and peridermal tissues. In September 2013, 10 symptomatic roots were collected from each of three fields in Jilin and Heilongjiang provinces. One piece of symptomatic skin tissue from each root was excised, surface-disinfested in 1% NaClO for 3 min, rinsed three times with sterile water, and then placed on tetracycline-amended (50 μg/ml) potato dextrose agar. After incubation at 22 ± 1°C in the dark for a week, small olivaceous black colonies developed from the symptomatic tissue from five of the 30 samples. No spores were observed. A single hyphal tip of each colony was transferred to a fresh V8 agar plate to purify the culture. Two-week-old colonies on V8 agar were olivaceous gray, and 42 to 46 mm in diameter with an outer white margin (3 to 5 mm wide). Conidia produced in V8 broth after 3 weeks with a 12-h photoperiod were straight and hyaline, cylindrical or subcylindrical with no or one septum. Mature conidia were 12.8 to 21.8 × 2.2 to 4.5 μm (mean 18.2 × 3.0 μm, n = 100 conidia for each of three isolates). Three isolates selected randomly were further identified by analyzing the partial sequences of the ITS region of rDNA with primers ITS4 and ITS5 (5), and partial sequences of β-tubulin with the primers tub2F and tub2R (1). Sequences of the three isolates (GenBank Accession Nos. KJ149287, KJ149288, and KJ149290 to 93) showed 99% to 100% homology with previously identified and deposited Rhexocercosporidium panacis isolates (DQ2499992 and DQ457119) for both loci (3). Therefore, the three isolates were identified as R. panacis and deposited in China General Microbiological Culture Collection Center (CGMCC3.17259 to 61). Pathogenicity of R. panacis in Asian ginseng was investigated using these three isolates as described previously with slight modifications (4). Bare roots of 3-year-old Asian ginseng were surface-disinfested as described above, and inoculated with mycelial plugs (4 mm diameter) cut from the margin of actively growing colonies of the isolates on V8 agar. Three mycelial plugs were placed on each root at 3-cm intervals and four roots (replicates) were inoculated for each isolate. Four additional roots were inoculated with non-colonized agar plugs as control. The treated roots were placed on moist filter paper in an enamel tray. The plates were sealed with plastic wrap to prevent desiccation and incubated in the dark at 18 ± 1°C. Four weeks post inoculation, all the inoculated ginseng roots showed red-brown lesions, which turned to dark red or black over time. R. panacis was recovered from symptomatic roots for all isolates and confirmed by ITS sequence analysis. The mock-inoculated control roots remained symptomless and no R. panacis was isolated. The inoculation experiment was repeated and showed the same results. R. panacis was reported in 2006 to infect roots of Panax quinquefolius (2,3,4). To our knowledge, this is the first report of R. panacis causing rusty root of P. ginseng.

Molecular identification and biodiversity of potential allergenic molds (Aspergillus and Penicilium) in the poultry house: first report

The production of poultry has proved to be a significant source of fungi. Penicillium and Aspergillus are among the most important species, because they can cause irritations, infections, allergies, spoilage of food and beverages and are able to produce dangerous mycotoxins. Detection and identification of the species in these genera is very significant, because it provides relevant information about the properties of the responsible strains. Therefore, their taxonomic was determined using a phenotypic and molecular (ITS and partial β-tubulin sequences) methods. Investigations were conducted in the poultry house located near Wroclaw in Lower Silesia (Poland). Using morphological analyses, 215 fungal strains were identified. Among them, 56 belonged to the Penicilium and Aspergillus genera. The results obtained from sequence analysis corresponded well with the morphological identification. Classification at species level was possible in most cases using the sequence data. Most isolates belong to Penicilium genus, with the follow dominating species (P. chrysogenum, P. polonicum and P. olsonii).

Identification and pathogenicity of Botryosphaeriaceae species associated with coast live oak (Quercus agrifolia) decline in southern California

Symptoms of decline have been observed on dying coast live oak (Quercus agrifolia) trees in areas throughout southern California that are both infested and uninfested by the gold-spotted oak borer (GSOB). The purpose of this study was to identify and assess the pathogenicity of several anamorph species of the Botryosphaeriaceae, including Diplodia corticola, Dothiorella iberica and Diplodia agrifolia sp. nov., that were recovered consistently from symptomatic tissues. Species were identified morphologically and by phylogenetic analyses of the complete sequence of the internal transcribed spacer (ITS) of the rDNA and partial sequences of β-tubulin and elongation factor (EF1-α) genes. Results from morphological assessments and phylogenetic analyses support the erection of a new species closely related to D. mutila, described herein as Diplodia agrifolia sp. nov. Pathogenicity of all species was verified by wound inoculation of 1 y old coast live oak seedlings under controlled conditions. Isolates of D. corticola were the most aggressive tested, and isolates of D. agrifolia were the second most aggressive. Both species caused bleeding symptoms on inoculated seedlings. Seedlings inoculated with D. corticola died within 4 wk, with the pathogen progressing up and down through the xylem in advance of living phloem and moving throughout the taproot in 70% of inoculated seedlings. Colonization and re-isolation was successful for all species. All three fungal species represent newly recorded fungal pathogens of coast live oak in California. Results from the pathogenicity test suggest that these fungi play a role in the decline of coast live oaks throughout southern California.