Partial Sequence Of Β-tubulin Gene Research Articles

First report of Leptosphaeria biglobosa 'brassicae' causing leaf spots in Eutrema japonicum in Yunnan, China.

Wasabi (Eutrema japonicum) is a root vegetable that is cultivated at large scales in southwestern China. In November 2021, approximately 40% of plants in a forested plantation in Dadishui, Yunnan Province, China (25.47°N, 103.22°E), showed leaf spot symptoms. The early symptoms were small black spots that gradually expanded into irregular brown to black lesions (0.5-1.5 cm), which were restricted by leaf veins. Yellow halos were observed at the outer edges of necrotic lesions. To identify the causal agent, we collected 20 diseased leaves and obtained fungal isolates from symptomatic leaf tissues. Following surface sterilization with 75% ethanol for 30 s, the tissues were cultured on potato dextrose agar (PDA) plates and incubated at 25°C under a 12 h light/12 h dark light cycle. After 7 days of incubation, a total of 12 isolates were obtained through single-spore culture. All isolates had similar colony morphology, and produced fluffy white mycelia and yellow pigment after 1 week of PDA culture at 25°C, and blackish- brown mycelium, tan pigment, and conidia after 2 weeks. The conidia were hyaline and cylindrical, with an average size of 4.6 μm × 2.2 μm. These morphological characteristics similar to the description of Leptosphaeria biglobosa (Shoemaker et. al, 2001) and Leptosphaeria maculans (Vincenot et al. 2008). Genomic DNA was extracted from mycelium of isolate SK-1, which was harvested from 10-day-old PDA culture using a FAST plant genomic DNA Extraction Kit (Biomed, China), following the manufacturer's instructions. The species-specific primers LbigF, LmacF, and LmacR (Liu et al. 2006) were used for identification via polymerase chain reaction (PCR). A 444-bp fragment characteristic of L. biglobosa 'brassicae' (Lbb), and a 330-bp of L. maculans 'brassicae' (Lmb) were amplified, respectively. Internal transcribed spacer (ITS) sequences (592 bp), part of the 5' end of beta-tubulin (968 bp), and actin (899 bp) were also amplified using the primers ITS1/ITS4, BT1/BT2, and ACTF/ACTR (Vincenot et al. 2008), respectively. PCR was performed in a volume of 25 μL containing 12.5 μL 2 × T5 Super PCR Mix (Tsingke Biotech, Beijing, China), 1 μL 10 μM primer (Tsingke Biotech), 1 μL DNA template, and an aliquot of sterile water to attain the total volume. The thermal cycler settings were 5 min at 98°C; 35 cycles of 10 s at 98°C, 10 s at 58°C, and 30 s at 72°C; and extension for 2 min at 72°C. The ITS sequence of isolate SK-1 (GenBank accession no. OQ216838), the partial β-tubulin gene sequence (OQ241183), and the actin gene sequence (OQ241184) indicated 100% query cover and 100% identity with L. biglobosa (DQ458906), Lbb strain B3.6 (AY748995), and Lbb strain 2379-4 (AY748949), respectively. Phylogenetic analysis (King et al. 2022) also identified of isolate SK-1 as Lbb. To determinate its pathogenicity, isolate SK-1 was grown on PDA incubated at 28°C for 2 weeks, and conidial suspensions were prepared at a concentration of 106 conidia/mL. Then, 15 leaves of 4-month-old E. japonicum seedlings were needle-wounded on the front and inoculated by syringe injection of 10 μL of the appropriate conidial suspension. We used 10 μL of the sterilized distilled water as the control under forest growth conditions. All inoculation sites were covered with cotton strips and moistened with 1.0 mL sterile water to maintain humidity. After 12 days of incubation, the leaves developed symptoms similar to those observed in the field, and the fungus was reisolated from diseased leaves, whereas the controls remained healthy. Based on these results, we identified L. biglobosa 'brassicae' as the causal agent of leaf spot on E. japonicum in China. This fungus has been reported to cause blackleg in many Brassica crops in China such as Brassica napus (Fitt et al. 2006), Brassica oleracea (Zhou et al. 2019), B. juncea var. tumida (Deng et al. 2020), Brassica rapa subsp. pekinensis (Yu et al. 2021). To the best of our knowledge, this is the first report of L. biglobosa causing leaf spots in E. japonicum in China. Our data provide a basis for disease management in E. japonicum production in China.

A SYBR Green qPCR assay for specific detection of Colletotrichum ocimi, which causes black spot of basil

Colletotrichum ocimi causes black spot of basil (Ocimum basilicum) and is a serious threat to basil cultivation as it compromises leaf production. The pathogen also infects seeds, which could become primary sources of inoculum for spread of black spot. A SYBR Green real-time PCR assay was developed to detect Colletotrichum ocimi in basil leaves and seeds, based on the partial β-tubulin (tub2) gene sequence. Two primer sets were designed and tested. The selected primer pairs produced amplicons of 130 bp. The real-time PCR assay was validated for analytical specificity, sensitivity, selectivity, repeatability and reproducibility. The assay was specific for C. ocimi with respect to ten Colletotrichum spp. and to another 12 pathogens of basil plants. Sensitivity was 1 pg µL-1 of genomic fungal DNA and amplification analyses were not influenced by basil genomic DNA. The assay detected and quantified C. ocimi in artificially inoculated basil leaves. This is the first specific primer set for C. ocimi, which allows rapid detection and quantification of the pathogen is a useful tool for diagnostics in plants. Detection in seeds would also be possible, but will require an optimized extraction method. The qPCR detection of C. ocimi in planta can contribute to adoption of effective preventive disease management strategies.

Enzyme activity of Aspergillus section Nigri strains isolated from the Korean fermentation starter, nuruk.

Aspergillus section Nigri is a fungus used industrially because of its ability to produce enzymes such as cellulolytic, amylolytic and proteolytic enzymes. In this study, we obtained twenty-eight strains of Aspergillus section Nigri from the traditional Korean fermentation starter, nuruk, which is known as a mixed culture of enzymatic filamentous fungi and yeasts. All strains were identified as Aspergillus section Nigri through combined phylogenetic analysis using partial β-tubulin and calmodulin gene sequences. The cellulase, amylase and protease activities of Korean strains were measured and compared with ten reference strains of Aspergillus niger. Most Korean strains showed higher cellulolytic activity than reference strains, and Aspergillus neoniger KCN5 showed the highest β-glucosidase activity. Two-thirds of the Korean strains showed similar levels of α- and glucoamylase activity as the reference strains. The protease activity of Aspergillus section Nigri strains was the highest at pH 3.0, and A. niger KSJ2 showed the highest acidic protease activity. By comparing ten reference strains and twenty-eight Korean strains, our results suggested useful Aspergillus section Nigri strains from nuruk with high enzyme activity, such as KCN5 and KSJ2, and their potential for industrial applications as enzyme producers.

Polyphasic taxonomic characterization of nine Penicillium species from soil of different parts of India

Aim: Morpho-molecular analyses for taxonomic characterization of nine predominant Penicillium species present in the soil of different parts of India. Methodology: Fifteen Penicillium isolates were isolated from the soil samples collected from the experimental field of ICAR-Indian Agricultural Research Institute (IARI), New Delhi. Another twenty-six isolates were procured from Indian Type Culture Collection (ITCC), Division of Plant Pathology, ICAR-IARI, New Delhi which were isolated from the soil of different parts of India. Total 41 isolates were characterized following distinct macroscopic (colony texture, colony colour exudate production; soluble pigmentation; reverse coloration and mycelial growth) and microscopic observations (type of penicillus; shape of phialides; conidial shape, size and pigmentation). Molecular characterization was done using partial β-tubulin gene sequence which is considered an excellent marker in differentiating Penicillium species. Results: The morphological data in species description perfectly matched with molecular data generated using β-tubulin gene marker and nine different species viz., P. aethiopicum, P. chrysogenum, P. crustosum, P. janthinellum, P. mononematosum, P. oxalicum, P. polonicum, P. singorense and Talaromyces pinophilus (Syn. P. pinophilum) were confirmed. Interpretation: Above nine Penicillium species are found predominantly in the soil collected from different parts of India. The β-tubulin gene can be considered as an excellent marker to differentiate Penicillium species as confirmed from this study. The combined morpho- molecular analyses can be further utilized to delineate morphologically similar Penicillium species and also helpful to establish new species of Penicillium.

Isolation and identification of Zalaria sp. Him3 as a novel fructooligosaccharides-producing yeast.

This study aimed at obtaining a novel fructooligosaccharides (FOS)-producing yeast, which was different from conventional FOS producers, Aureobasidium spp. Strain Him3 was newly isolated from a Japanese dried sweet potato as a FOS producer. The strain exhibited yeast-like cells and melanization on the potato dextrose agar medium, and formed very weak pseudomycelia on the yeast extract polypeptone dextrose agar medium. Based on the internal transcribed spacer (ITS) region of ribosomal DNA and a partial β-tubulin gene sequences, the strain Him3 was identified as Zalaria sp. The β-fructofuranosidase (FFase) produced by strain Him3 was localized on the cell surface (CS-FFase) as well as in the culture broth (EC-FFase). The FOS production yields by CS-FFase and EC-FFase from 50% sucrose were 63.8% and 64.6%, respectively, to consumed sucrose after the reaction for 72h. We successfully isolated a novel black yeast, Zalaria sp. Him3, with effective capacity for FOS production. Phylogenetic analysis revealed that strain Him3 was distantly related with the conventional FOS producers, Aureobasidium spp. Since FFase of strain Him3 demonstrated high production yields of FOS, it could be applied to novel industrial production of FOS, which is different from conventional methods.

The polyphasic re-identification of a Brazilian Aspergillus section Terrei collection led to the discovery of two new species

Species belonging to Aspergillus section Terrei have a worldwide distribution and are phenotypically similar. Most species grow either moderately or fast on agar media, and their conidial colors are often in shades of cinnamon brown. In the present study, 79 section Terrei strains stored at the University Recife Mycologia (URM) Culture Collection (Micoteca URM) in Brazil were re-identified using a polyphasic approach, combining partial β-tubulin (BenA) and calmodulin (CaM) gene sequences and morphological data. Seven known (A. terreus, A. pseudoterreus, A. alabamensis, A. citrinoterreus, A. hortai, A. neoafricanus, and A. niveus) and two new species, A. barbosae and A. recifensis, were present among the studied strains. The new species were described based on internal transcribed spacer (ITS), BenA, CaM, and RNA polymerase II second largest subunit (RPB2) sequences in combination with morphological features and extrolite profiles; the most important characteristics for distinguishing the new from the accepted section Terrei species are given. Aspergillus recifensis produces the mycotoxin citreoviridin and A. barbosae citrinin.

First Report of Trichothecium roseum Causing Pink Fruit Rot of Prunus davidiana in China

Pink fruit rot of peach (Prunus davidiana) was observed in an orchard near Huairou, Beijing, China, in 2018. Approximately 90% of peach trees were infected, and diseased fruits were covered with pink-colored mold. To identify the causal agent, diseased fruits were collected, and fungal isolates were obtained from symptomatic fruit tissue. Initially, on potato dextrose agar, the isolates produced thin mycelium, with rare aerial hyphae at 28°C. After 3 days of incubation, the mycelium became pink and produced numerous powdery conidia. Conidia were pyriform, ovate, with papillary protuberances at one end. They were colorless individually but pinkish in mass, with one septation that slightly constricted the middle of the spore. The conidia had an average size of 15 × 6 μm under a light microscope. Two isolates, HB1-1a and HB1-2c, were used in the pathogenicity tests. Each isolate was inoculated onto 10 healthy P. davidiana fruit by pipetting with 100 μl of spore suspension at the concentration of 1 × 10⁶ conidia/ml. Distilled water served as the control. All treated fruits were enclosed in a plastic bag and stored at 28°C under a 12-h light/dark cycle. After 15 days of incubation, symptoms similar to those observed in the orchard developed. The same fungus was reisolated from the diseased fruit. To further confirm the identity of the pathogen, the internal transcribed spacer (ITS) and part of the 5′ end of the β-tubulin (TUB) genes of the isolates HB1-1a and HB1-2c were amplified by using two different primer sets: ITS1/ITS4 (White et al. 1990) and T1/T22 (Hamid et al. 2014). The PCR products had sizes of approximately 700 bp for ITS and 1.5 kb for TUB. The ITS sequence of isolates HB1-1a and HB1-2c (GenBank nos. MN629234 and MN629235) indicated 99.67% identity with Trichothecium roseum (GenBank no. EU552162.1). The partial β-tubulin gene sequence (GenBank nos. MN640076 and MN640077) showed a 100% homology with T. roseum strain UoS09 (GenBank no. KJ607590.1). Therefore, this fungus was identified as T. roseum based on morphological characteristics and molecular data. T. roseum is reported to have a wide range of host (Hong et al. 1997; Inacio et al. 2011). In China, this fungus was first to be found in apple fruit introduced by a traveler in 2018 (Xu et al. 2018). To our knowledge, this is the first report of T. roseum causing pink fruit rot of P. davidiana in China.

Five New Records of the Family Aspergillaceae in Korea, Aspergillus europaeus, A. pragensis, A. tennesseensis, Penicillium fluviserpens, and P. scabrosum

During an investigation of the fungi from the Aspergillaceae family obtained from different environmental sources in Korea, we isolated six strains, including CNUFC WJC9-1, CNUFC BPM36-33, CNUFC MSW6, CNUFC ESW1, CNUFC TM6-2, and CNUFC WD17-1. The morphology and phylogeny of these isolates were analyzed based on their partial β-tubulin (BenA) and calmodulin (CaM) gene sequences. Based on the morphological characteristics and sequence analyses, the isolates CNUFC WJC9-1, CNUFC BPM36-33, CNUFC TM6-2, and CNUFC WD17-1 were identified as A. europaeus, A. pragensis, Penicillium fluviserpens, and P. scabrosum, respectively, and isolates CNUFC MSW6 and CNUFC ESW1 were identified as A. tennesseensis. To the best of our knowledge, the species A. europaeus, A. pragensis, A. tennesseensis, P. fluviserpens, and P. scabrosum have not been previously reported in Korea.

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.

Diaporthe limonicola Causing Leaf Spot Disease on Areca catechu in China

The areca palm (Areca catechu L.), belonging to the Palmaceae family, is an important economic crop in Hainan. In June 2018, symptoms of leaf spot were observed on nearly 20% of A. catechu in Qionghai, Hainan (19°40′N; 110°33′E). At first, leaves exhibited small yellow spots; as symptoms progressed, the middle of the lesions appeared black with distinct yellow halos. Lesions were long oval, sometimes irregular with black to brown, small black spots and distinct yellow halos. As the lesions continued to expand, necrotic spots enlarged, gradually gray, and then combined to form larger necrotic areas. Ten leaves with typical symptoms were randomly collected. Small tissue sections (5 × 5 mm) were excised from the margins of lesions, disinfected with 75% ethanol for 10 s and 1% sodium hypochlorite for 2 min, followed by a triple wash with sterile water, plated on PDA, and incubated at 28°C. Three days after incubation, fast-growing fungal colonies with white mycelia appeared. Incubation continued for 3 weeks and few pycnidia developed. Alpha conidia were 5.9 to 8.5 × 2.3 to 3.0 µm (avg. 7.3 × 2.7 µm, n = 100), aseptate, hyaline, fusiform, and acute at both ends. Beta conidia were 14.8 to 29.6 × 1.3 to 2.1 µm (avg. 22.5 × 1.7 µm, n = 100), hyaline, filiform, curved, and tapering toward both ends. These morphological characteristics were similar to Diaporthe spp. (Guarnaccia and Crous 2017). To further confirm the identity of the isolate, single-spore isolates were cultured on PDA and selected for DNA extraction. The internal transcribed spacer (ITS) gene was amplified using primers ITS1 and ITS4 (White et al. 1990), a partial sequence of β-tubulin (TUB) gene by T1 (O’Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995), translation elongation factor 1-α (TEF1) gene by EF1-728F/EF1-986R, and calmodulin (CAL) by CAL-228F/CAL-737R (Carbone and Kohn 1999); sequence data were deposited in GenBank (MN424525, MN424539, MN424567, MN424581). BLAST analysis demonstrated that these sequences were 99% similar to the ITS (MF418423), TUB (MF418583), TEF1 (MF418502), and CAL (MF418257) of D. limonicola. Moreover, a phylogenetic tree was constructed using the method of maximum likelihood (MEGA7.0 (Kumar et al. 2016)) with a combined dataset of ITS, TUB, TEF1, and CAL sequences, which clustered the isolate with D. limonicola (CPC 31137) with high bootstrap support (100%). Based on the morphology, sequence data, and phylogenetic analysis, these isolates were determined as D. limonicola. To validate the results, a pathogenicity test was performed. Ten healthy leaves were washed with sterile water, and each leaf was slightly punctured eight times by a sterile pin and divided into two groups. The first group was placed on a mycelial disk taken from the edges of 4-day-old PDA cultures on each wounded leaf, whereas the other group was placed on PDA agar as a control. All plants were incubated at 28 ± 2°C and 100% relative humidity. Seven days after inoculation, leaves placed on the mycelial disk exhibited the typical symptoms (i.e., small black spots and gray to brown lesions surrounded by yellow halos), whereas controls showed no symptoms. Further, D. limonicola was reisolated from the leaf spot. Pathogenicity tests were repeated thrice with the same results. Koch’s postulate was achieved by the reisolation of D. limonicola from the inoculated leaves. D. limonicola has previously been reported to cause trunk canker of Citrus limon and C. sinensis in Italy (Guarnaccia and Crous 2017). To our knowledge, this is the first report of D. limonicola causing leaf spot on A. catechu in China.

First Report of Shoot Dieback on Apple Caused by Diaporthe nobilis in China

In May 2017, apple trees (Malus pumila Mill.) with shoot dieback were observed in Laiyang (36.97°N, 120.71°E), Shandong Province, China. Disease symptoms included twig and shoot dieback and black necrotic lesions. In the later stage, numerous black pycnidia were observed on the surface of necrotic bark. Small pieces (4 to 5 mm) of necrotic tissues from different apple trees were disinfected for 1 min in 75% ethanol, rinsed thrice with sterile distilled water, and plated onto potato dextrose agar (PDA) with 0.05% lactic acid at 25°C. Eight isolates were obtained, and colonies from all isolates were white. Abundant pycnidia developed on PDA for 3 weeks at 25°C, which produced α-conidia measuring 6.2 to 8.9 × 2.2 to 3.2 μm and β-conidia measuring 18.5 to 27.6 × 1.1 to 2.0 μm (n = 50). α-Conidia were one-celled, hyaline, fusiform, and biguttulate, whereas β-conidia were one-celled, hyaline, and filiform. To identify these isolates to species, total DNA was extracted from the monoconidial isolates. The internal transcribed spacer region (ITS), a partial sequence of β-tubulin gene (TUB), translation elongation factor 1-α gene (TEF1), and calmodulin (CAL) were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), EF1-728F/EF1-986R, and CAL-228F/CAL-737R (Carbone and Kohn 1999), respectively. The sequences (GenBank accession nos. MK522125 for ITS, MK540236 for TUB, MK540238 for TEF1, and MK540240 for CAL) were aligned using BLASTn in GenBank, showing 99.4 to 100% with the sequences of ITS (KC343147), TUB (KC344120), TEF1 (KC343872), and CAL (KC343388) from the ex-epitype strain of Diaporthe nobilis complex (Gomes et al. 2013). The identity of the isolates was proved by maximum likelihood and Bayesian inference with a combined dataset of ITS, TUB, TEF1, and CAL sequences. The isolates were nested within the clade that includes D. nobilis with 98% bootstrap support and 1.0 Bayesian inference. The pathogenicity of three isolates (SDAp1, SDAp2, and SDAp3) was evaluated on 2-year-old shoots using a wound-inoculation method on living apple trees (cv. ‘Fuji’). Fifteen shoots were wounded (3 mm diameter, 2 mm deep) with a scalpel by cutting into the bark and inoculated with 10 μl of spore suspension (10⁶ α-conidia/ml). Wounds were then sealed with Parafilm. The same number of wounded shoots was inoculated with sterile water as a control. The pathogenicity test was conducted three times independently. Necrotic lesions (2.8 ± 0.5 cm in length) developed around the inoculation points after 3 weeks of inoculation. No disease symptoms developed on the control shoots. The pathogen was successfully reisolated from 93.3% of the inoculated shoots. In China, D. nobilis has been reported as a new pathogen of postharvest rot of blueberry (Yu et al. 2018) and shoot canker of chestnut (Zhang et al. 2018). To our knowledge, this is the first report of D. nobilis causing shoot dieback on apple in China. This report is important for the new research aiming at management of apple shoot disease.

First Report of Lasiodiplodia pseudotheobromae Causing Canker and Shoot Dieback on Apple in China

In July 2017, apple trees (Malus pumila Mill.) with canker and shoot dieback symptoms on side branches were observed in several young orchards in Yantai, Shandong Province, China. Symptoms included shoot dieback, discoloration of xylem tissues, cankers, and sunken necrotic bark lesions, which surrounded the branches and resulted in the death of the branches. The disease is symptomatically different from canker diseases of apple earlier described. Small pieces (4 to 5 mm) of necrotic tissues from the shoot cankers were disinfected for 1 min in 75% ethanol, rinsed thrice with sterile distilled water, and plated onto potato dextrose agar (PDA) with 0.05% lactic acid. White colonies developed from symptomatic tissues but became dark gray mycelia with age. Pycnidia were produced on sterilized apple twigs on 2% water agar after incubation for 3 weeks at 25°C. Conidia were unicellular, ellipsoidal, initially hyaline, and became dark brown and developed a central septum with age. Conidia measured 21.3 to 31.9 × 12.4 to 16.9 µm (n = 50). On the basis of morphological characteristics, the isolates were identified as Lasiodiplodia sp., a member of the Botryosphaeriaceae family (Alves et al. 2008; Phillips et al. 2013). For further identification, total DNA was extracted from the mycelium of three monoconidial isolates (La1, La2, and La3). The rDNA internal transcribed spacer (ITS), a partial sequence of β-tubulin gene (TUB), and translation elongation factor 1-α gene (TEF1) were amplified and sequenced using primers ITS1/ITS4, Bt2a/Bt2b, and EF1-688F/EF1-1251R, respectively (Alves et al. 2008; Glass and Donaldson 1995; White et al. 1990). The sequences (GenBank accession nos. MK408640 to MK408642 for ITS, MK446722 to MK446724 for TUB, and MH829861 to MH829863 for TEF1) were aligned using BLASTn in GenBank, showing 99 to 100% homology with the sequences of ITS (EF622077), TUB (EU673111), and TEF1 (EF622057) from L. pseudotheobromae isolate CBS 116459 (Alves et al. 2008). The pathogenicity testing of three isolates was conducted using the wound inoculation method both on living apple trees and detached twigs. Thirty 2-year-old twigs were wounded with a scalpel on the phloem tissues and inoculated by placing 5-mm² agar plugs containing 2-day-old cultured hyphae or 10 μl of an inoculum suspension (10⁵ conidia/ml), which were then sealed with Parafilm. The same number of wounded twigs was inoculated with uncolonized PDA plugs or sterile water as controls. After 7 days, necrotic lesions (35 to 60 mm in diameter) developed around the inoculation points, which resembled the symptoms on infected apple shoots in the field. No symptoms were observed on the control twigs, and the pathogenicity test was conducted thrice. The pathogen was reisolated from symptomatic twigs and was reidentified as L. pseudotheobromae on morphological and molecular analysis. Although L. pseudotheobromae was previously reported on several forest and fruit trees (Alves et al. 2008; Awan et al. 2016), to our knowledge, this is the first report of the pathogen causing canker and shoot dieback on apple in China. This report is important for the new research aiming at management of apple canker disease.

Species diversity in Penicillium and Talaromyces from herbivore dung, and the proposal of two new genera of penicillium-like fungi in Aspergillaceae.

Coprophilous fungi are saprotrophic organisms that show great diversity, mainly on herbivore dung. The physico-chemical characteristics of this peculiar substrate combined with the high level of fungal adaptation to different environmental conditions offer the perfect setting for discovering new taxa. This study focused on the species diversity of penicillium-like fungi isolated mainly from herbivore dung collected at different Spanish locations. From 130 samples, a total of 104 isolates were obtained, and 48 species were identified. Preliminary identifications were based on morphology and partial β-tubulin (tub2) gene sequences. Putative new taxa were characterized by a multi-gene sequencing analysis testing the tub2, the internal transcribed spacer rDNA (ITS), calmodulin (cmdA), and RNA polymerase II second largest subunit (rpb2) genes, and a detailed phenotypic study. Using this polyphasic approach and following the genealogical concordance phylogenetic species recognition (GCPSR) method, we propose the new genera Penicillago (for Penicillium nodositatum) and Pseudopenicillium (for Penicillium megasporum and P. giganteum) in the family Aspergillaceae, and 11 new species, including seven Penicillium, three Talaromyces and one Pseudopenicillium. A lectotype and epitype are designed for Penicillium nodositatum. Our results show that the species diversity of penicillium-like fungi on herbivore dung has not been widely studied and that this substrate seems to be a good reservoir of interesting Eurotialean fungi.

The Molecular Identification and Antifungal Susceptibilities of Aspergillus Species Causing Otomycosis in Tochigi, Japan

Aspergillus species are the most common pathogenic fungi involved in otomycosis, an infection of the outer ear canal. In this study, we examined the incidence of Aspergillus infections and the antifungal susceptibilities of 30 Aspergillus species isolates from patients with otomycosis who visited Saiseikai Utsunomiya Hospital between August 2013 and July 2016. Based on the morphological test results, the strains were identified as Aspergillus niger sensu lato (20 strains), A. terreus sensu lato (7 strains), and A. fumigatus sensu lato (3 strains). In contrast, the molecular identifications based on analyzing the isolates' partial β-tubulin gene sequences revealed them to be A. niger sensu stricto (12 strains), A. tubingensis (8 strains), A. terreus sensu stricto (7 strains), and A. fumigatus sensu stricto (3 strains). The antifungal susceptibility test results indicated that strains of A. tubingensis and A. niger sensu stricto displayed lower susceptibilities to ravuconazole, compared with the other isolates. The Aspergillus strains from this study showed low minimum inhibitory concentrations toward the azole-based drugs efinaconazole, lanoconazole, and luliconazole. Therefore, these topical therapeutic agents may be effective for the treatment of otomycosis.

First Report of Diaporthe nobilis Causing Postharvest Rot of Blueberry in Shandong Province, China

Blueberry (Vaccinium L.) has become increasingly popular in China over the last several years owing to its high nutritional value. During June 2015, a postharvest fruit rot was observed on blueberry cultivar Duke cultivated in Qingdao, Shandong, China, and stored 7 days at 4°C after harvesting. Infected fruits showed soft and rotted lesions, covered with grayish or white mycelium under high humidity. To identify the causal agent of the disease, tissues were excised from surface-sterilized fruits and cultured on potato dextrose agar (PDA). Fungal colonies developed from all pieces of diseased tissues. Most isolates were identified morphologically and molecularly as Botrytis cinerea, Penicillium expansum, and Alternaria alternata, the important postharvest fungal pathogens of blueberry in China (Dai et al. 2016). Seven previously unidentified isolates were obtained, and all had white mycelia on PDA. After incubation for 3 weeks at 25°C on PDA, abundant pycnidia developed and produced hyaline, fusiform, biguttulate α-conidia and one-celled, hyaline, filiform β-conidia. The α-conidia dimensions were 5.9 to 8.7 × 2.0 to 3.4 μm (average 7.3 × 2.6 μm) and β-conidia were 16.5 to 27.8 × 0.9 to 1.9 μm (average 22.3 × 1.3 μm, n = 50). Morphological characteristics suggested the identity of the fungal isolates to be Diaporthe nobilis. To confirm identification, DNA was extracted from the mycelium of three representative isolates (SDB1, SDB2, and SDB3). The rDNA internal transcribed spacer (ITS), a partial sequence of β-tubulin gene (TUB), translation elongation factor 1-α gene (TEF1), and calmodulin (CAL) were sequenced using primers ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), EF1-728F/EF1-986R, and CAL-228F/CAL-737R (Carbone and Kohn 1999), respectively. The consensus sequences (GenBank accession nos. MG557846 for ITS, MG557847 for TUB, MG557848 for TEF1, and MG557849 for CAL) showed highly similar (99, 100, 99, and 99%) with the sequences of ITS (KJ609006), TUB (KC344121), TEF1 (KC343874), and CAL (KC343395) from D. nobilis (Gomes et al. 2013). Pathogenicity tests were performed on 120 mature surface-disinfested blueberry (cv. Duke) fruits with a conidial suspension (10⁶ α-conidia/ml) of the three isolates. Sixty fruits were stab-inoculated to a depth of 3 mm using a sterile needle. For each isolate, 20 wounded and 20 unwounded fruits were inoculated with a 10-μl droplet. Forty fruits with or without wounding were treated with sterile water to create controls. Fruits were incubated in a chamber with a 12-h photoperiod at 25°C and 80% humidity. After 7 days, typical symptoms of postharvest rot with softened areas 3.7 to 6.4 mm in diameter were observed at wound sites. Experiments were conducted three times, and the pathogen was reisolated each time and confirmed as D. nobilis based on morphological and molecular analysis. Noninoculated and nonwounded fruits showed no symptoms. To our knowledge, this is the first report of D. nobilis causing blueberry postharvest rot in China. To prevent the spread of blueberry rot diseases, more care should be taken to avoid wounding during or after harvest.

Genetic and Toxigenic Variability within Aspergillus flavus Population Isolated from Maize in Two Diverse Environments in Kenya.

Aspergillus flavus is the main producer of carcinogenic aflatoxins in agricultural commodities such as maize. This fungus occurs naturally on crops, and produces aflatoxins when environmental conditions are favorable. The aim of this study is to analyse the genetic variability among 109 A. flavus isolates previously recovered from maize sampled from a known aflatoxin-hotspot (Eastern region, Kenya) and the major maize-growing area in the Rift Valley (Kenya), and to determine their toxigenic potential. DNA analyses of internal transcribed spacer (ITS) regions of ribosomal DNA, partial β-tubulin gene (benA) and calmodulin gene (CaM) sequences were used. The strains were further analyzed for the presence of four aflatoxin-biosynthesis genes in relation to their capability to produce aflatoxins and other metabolites, targeting the regulatory gene aflR and the structural genes aflP, aflD, and aflQ. In addition, the metabolic profile of the fungal strains was unraveled using state-of-the-art LC-MS/MS instrumentation. The three gene-sequence data grouped the isolates into two major clades, A. minisclerotigenes and A. flavus. A. minisclerotigenes was most prevalent in Eastern Kenya, while A. flavus was common in both regions. A. parasiticus was represented by a single isolate collected from Rift Valley. Diversity existed within the A. flavus population, which formed several subclades. An inconsistency in identification of some isolates using the three markers was observed. The calmodulin gene sequences showed wider variation of polymorphisms. The aflatoxin production pattern was not consistent with the presence of aflatoxigenic genes, suggesting an inability of the primers to always detect the genes or presence of genetic mutations. Significant variation was observed in toxin profiles of the isolates. This is the first time that a profound metabolic profiling of A. flavus isolates was done in Kenya. Positive associations were evident for some metabolites, while for others no associations were found and for a few metabolite-pairs negative associations were seen. Additionally, the growth medium influenced the mycotoxin metabolite production. These results confirm the wide variation that exists among the group A. flavus and the need for more insight in clustering the group.

First Report of Root Rot Caused by Calonectria ilicicola on Blueberry in Yunnan Province, China

Blueberry (Vaccinium spp.) has been cultivated in more than 20 provinces in China as reported in 2015. In July 2016, a root rot disease of blueberry causing plant death was found in Qujing (24°49′N; 103°59′E), Yunnan Province. Approximately 5% of plants in the field showed symptoms characterized by leaf chlorosis, black rot on roots and stems, and finally the whole plant wilted in about 20 days. In order to identify the causal agent of this disease, typical disease samples were collected. Twenty infected root tissues were sterilized and put on potato dextrose agar (PDA) at 25°C (Fang 1998) and nine fungal isolates were obtained. The isolate YNQJ01Aa was chosen for further testing. Its colony on PDA was white to pale yellow. The conidia were collected from SNA colony after growing for 10 days. Conidia presented with one to three septa (mostly one septa). Fifty conidia from one isolate measured 59.37 to 71.33 × 5.93 to 7.91 μm (avg. 62.25 × 6.22 µm). Ascospores were produced in perithecia on PDA and were 30.16 to 38.89 × 5.78 to 7.76 µm (avg. 34.97 × 6.52 µm). By DNA sequencing of the internal transcribed spacer (ITS) rDNA region (GenBank accession no. MF785081), the identity of the isolate (YNQJ01Aa) was confirmed to be the genus Calonectria (99% homologous to several Calonectria spp. strains). The partial β-tubulin (TUB) gene sequence was also analyzed (MF785082) and the result of a BLAST search showed that it was 100% homologous to C. ilicicola strain (CBS 190.50). Further species identification was analyzed by phylogenic tree based on TUB (partial β-tubulin gene, T1/Bt2b), tef1 (translation elongation factor 1 alpha gene, EF1-728F/EF1-986R), and HIS3 (histone 3 gene, H3-1a/H3-1b) (Chen et al. 2011; Crous 2002). Sequences obtained were deposited in GenBank (TUB: MF785082, tef1: MF785083, HIS3: MF785084). Alignments were made using CLUSTAL X v.2.0, and a maximum likelihood phylogram was generated by MEGA v.7.0. Isolate YNQJ01Aa clustered together with C. ilicicola reference strain CBS190.50 (98% bootstrap). Three healthy 1-year-old ‘O’Neal’ blueberry plants were used to conduct pathogenicity tests by wound inoculation. Each plant was inoculated with 10 ml of conidial suspension (1 × 10⁵ conidia/ml) by the root irrigation method. Three plants were inoculated with the same amount of sterile water as control. All blueberry plants were incubated at 25°C. After 20 days, the test plants showed typical symptom of black root rot and finally the whole plant wilted and died, while the control plants remained healthy. The same colonial fungus was reisolated. This is the first report of C. ilicicola (anamorph Cylindrocladium parasiticum) infecting blueberry in China. Root rot disease can be difficult to detect early and therefore it is important to identify the etiology to develop appropriate disease management strategies. This fungus was previously reported on Medicago sativa in China (Pei et al. 2015).

First report of chilli anthracnose caused by Colletotrichum karstii in India

Chilli, Capsicum annum, is an annual herbaceous vegetable and spice grown in almost all the states of India. Anthracnose (fruit rot) of chilli caused by Colletotrichum species results in both pre- and post-harvest fruit decay with yield losses of up to 50% (Pakdeevaraporn et al., 3). The different species of Colletotrichum causing chilli anthracnose reported from India include: Colletotrichum acutatum, C. capsici, C. coccodes, C. dematium, C. gloeosporioides and C. siamense. In August 2014, chilli fruits showing typical anthracnose symptoms of sunken necrotic tissues with concentric brown black rings of acervuli were obtained from a farmer's field in Jalna, Maharashtra, India. Small pieces of necrotic tissue were examined under a microscope and spores inoculated on potato dextrose agar (PDA) containing 50 mg/l streptomycin sulphate. The plates were incubated at 28°C for 5 to 7 days and a pure culture of the Colletotrichum isolate was obtained by subculturing on fresh PDA plates. The colonies had white aerial mycelia with an orange conidial mass (Fig. 1). The colour of the colony on the reverse side was light orange. The mycelial growth rate on the PDA plate at 28°C and a 16/8 h light/dark cycle respectively, was 11 mm per day. Conidia were single celled, cylindrical with rounded base and apex. Mean length and width of conidia was 13.11 ±1.22 μm and 6.2 ±0.34 μm, respectively (Fig. 2). Molecular characterization of the Colletotrichum isolate was based on ITS rDNA and partial β-tubulin gene sequence comparisons. PCR amplification was done using the universal primer pair ITS 4/5 (ITS region of the nuclear ITS1-5.8S - ITS2 rDNA; White et al., 5) and β-tubulin gene Bt2a/b primers (Glass et al., 1). Both sequences were deposited in GenBank (Accession Nos. KX492583 and KX492584, respectively) and aligned with published sequences using MEGA version 6.0 (Tamura et al., 6), and phylogenetic analysis was done (Fig. 3). Blast searches in the NCBI database revealed that the ITS and β-tubulin gene sequences had 98% and 99% identity to Colletotrichum karstii (KT284369 and KC293650, respectively) confirming that the isolate obtained from infected chilli is C. karstii. To confirm pathogenicity, chilli fruits and seedlings were wounded with a sterile syringe and inoculated with 10 μl of a conidial suspension (c. 105 conidia/ml). Sterile water was used as a control. Inoculated fruits and seedlings were kept in a chamber at 28°C with 90% humidity. After seven days typical anthracnose symptoms were observed on chilli fruits and leaves (Fig. 4). This demonstrated that C. karstii re-isolated from symptomatic chilli fruits was able to cause anthracnose, thereby fulfilling Koch's postulates. Colletotrichum karstii as a causal agent of chilli anthracnose has been reported in Brazil (Lima et al., 2) and China (Wang et al., 4). To our knowledge, this is the first report of chilli anthracnose caused by C. karstii in India.

They seldom occur alone

Species of Coleophoma have been reported as plant pathogenic, saprobic or endophytic on a wide host range. The genus is characterised by having pycnidial conidiomata, phialidic conidiogenous cells intermingled among paraphyses, and cylindrical conidia. Coleophoma has had a confusing taxonomic history with numerous synonyms, and its phylogeny has remained unresolved. The aim of the present study was to use a polyphasic approach incorporating morphology, ecology, and molecular data of the partial large subunit of nrDNA (LSU), the internal transcribed spacer region with intervening 5.8S nrDNA (ITS), partial β-tubulin (tub2), and translation elongation factor 1-alpha (tef1) gene sequences to resolve its taxonomy and phylogeny. Based on these results the genus was found to be polyphyletic, with taxa tentatively identified as Coleophoma clustering in Dothideomycetes and Leotiomycetes. Species corresponding to the concept of Coleophoma s.str. (Dermateaceae, Helotiales, Leotiomycetes) were found to form a distinct clade, with five new species. Furthermore, Coleophoma was found to be linked to the newly established sexual genus, Parafabraea, which is reduced to synonymy. Isolates occurring on Ilex aquifolium in the Netherlands also clustered in Dermateaceae, representing a novel genus, Davidhawksworthia. In the Dothideomycetes, several taxa clustered in Dothiora (Dothideaceae, Dothideales), which is shown to have Dothichiza and Hormonema-like asexual morphs, with four new species. Furthermore, Pseudocamaropycnis is introduced as a new genus (Mytilinidiaceae, Mytilinidiales), along with Briansuttonomyces (Didymellaceae, Pleosporales) and Dimorphosporicola (Pleosporaceae, Pleosporales).

Penicillium excelsum sp. nov from the Brazil Nut Tree Ecosystem in the Amazon Basin'.

A new Penicillium species, P. excelsum, is described here using morphological characters, extrolite and partial sequence data from the ITS, β-tubulin and calmodulin genes. It was isolated repeatedly using samples of nut shells and flowers from the brazil nut tree, Bertolletia excelsa, as well as bees and ants from the tree ecosystem in the Amazon rainforest. The species produces andrastin A, curvulic acid, penicillic acid and xanthoepocin, and has unique partial β-tubulin and calmodulin gene sequences. The holotype of P. excelsum is CCT 7772, while ITAL 7572 and IBT 31516 are cultures derived from the holotype.