Partial Actin Research Articles

First Report of Anthracnose on Bletilla striata Caused by Colletotrichum fructicola in China.

Bletilla striata (Thunb.) Rchb. f. (Orchidaceae) is a traditional Chinese medicinal plant. In April 2018 and 2019, a leaf spot disease was observed on ∼20% of B. striata plants in two fields (∼1.4 h) in Guilin, Guangxi Province, China (Fig.1 A). Small, circular, brown spots were initially observed on the leaf surfaces, which progressively expanded into large, sunken, dark brown, necrotic areas. As the disease progressed, lesions merged into large, irregular spots, ultimately resulting in abscission. To determine the causal agent, small pieces (5 mm x 5 mm) were collected from the infected leaf tissues (n = 18), surface sterilized in 1% NaOCl for 2 min, and rinsed three times with sterile water. Then, the tissues were placed on potato dextrose agar (PDA) with chloramphenicol (0.1 g/L) and incubated under 12 h photoperiod at 26°C for 3 days. Seventeen isolates were obtained, of which twelve isolates with similar morphological characteristics were obtained from the germinated spores on PDA. Seven-day-old colonies on PDA appeared cottony, pale white to pale gray from above, and grayish-green from below. Conidia of strain BJ-101.3 were hyaline, aseptate, straight, and cylindrical, with rounded ends (Fig.1 E-G), measuring 11.3 to 15.9 μm × 4.0 to 6.4 μm (n = 50). Appressoria were brown to dark brown, with different shapes and a smooth edge (Fig.1 H-I), measuring 6.3 to 10.0 μm × 4.1 to 8.0 μm (n = 50). Morphological features were similar to C. gloeosporioides species complex (Weir et al. 2012, Fuentes-Aragón et al. 2018). For molecular identification, DNA was extracted from two isolates BJ-101.3 and BJ-101.13, following the CTAB method (Guo et al. 2000). The internal transcribed spacer (ITS) region, partial actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), manganese superoxide dismutase (SOD2), beta-tubulin (TUB2), glutamine synthetase (GS), and Apn2-Mat1-2 intergenic spacer and partial mating-type (ApMat) genes were amplified by PCR and sequenced (Weir et al. 2012, Silva et al. 2012, Vieira et al. 2017). The obtained sequences were deposited in GenBank (MW386818, MW386819, MW403508 to MW403519, and MW888410 to MW888413). BLASTN analysis of the obtained sequences showed 99% identity with those of C. fructicola (JX010165，JX010033, FJ917508, FJ907426, JX009866, JX010095, JX010327, JX010405, JQ807838) (Weir et al. 2012, Liu et al. 2015). A phylogenetic tree based on the concatenated sequences confirmed the isolates as C. fructicola (Fig.2). Furthermore, pathogenicity tests were conducted on six 1.5-year-old B. striata plants. Healthy leaves on the plants were inoculated with the conidial suspensions (106 conidia/mL; 10 μL) of the strains BJ-101.3 and BJ101.13. The conidial suspension of each isolate was inoculated onto at least three leaves. Another three plants inoculated with sterile water served as the control. All plants were covered with transparent plastic bags and incubated in a greenhouse at 26°C for 14 days with a 12 h photoperiod. Nine days post-inoculation, the inoculated leaves showed leaf spot symptoms, while the control plants remained symptomless (Fig.1 B-C). The experiments repeated three times showed similar results. Finally, C. fructicola was consistently reisolated from the infected leaves and confirmed by morphology and sequencing, fulfilling Koch's postulates. The outcome of this study will help in developing effective management measures against anthracnose of B. striata.

First Report of Colletotrichum fructicola Causing Anthracnose on Camellia yuhsienensis Hu in China.

Camellia yuhsienensis Hu is an endemic species from China, where is the predominant oilseed crop due to its anthracnose resistance (Kuang 2015; J. Li et al. 2020; Nie et al. 2020). In April 2019, anthracnose symptoms were observed on C. yuhsienensis in a plantation in Youxian, Zhuzhou, Hunan Province, China (113.32°E, 26.79°N). It was detected approximately 10% anthracnose incidence in 500 two-year-old plants in a 5000 m2 cultivated area. Diseased leaves showed irregular grayish brown spots with dark brown edges and dark brown undersides. Symptomatic tissues (4 to 5 mm2) were surface-disinfected for 90 s in 75% ethanol, then rinsed twice with sterile water, and finally incubated on PDA (potato dextrose agar) at 28℃ (Jiang et al. 2018). Pure cultures were obtained by the single-spore isolation method. A total of 100 fungal isolates were obtained from 85 symptomatic leaves, from which 81 had similar colony morphology. Colonies on PDA were white, fluffy and cottony, and becoming dark gray after 5 days. The character of the reverse of the colony were similar to that of the upper of the colony, but the color was darker at the same time. The isolates produced a large number of single-celled, hyaline, straight and cylindrical conidia, with 10.35 to 17.58 length × 3.46 to 5.69 μm width (x=13.61 × 4.63 μm, n = 30). The isolates were preliminarily identified as Colletotrichum spp. according to morphological features (Weir et al. 2012). Representative isolate YX2-5-2 was used for molecular identification: internal transcribed spacer (ITS), partial actin (ACT), chitin synthase (CHS-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genomic DNA regions were amplified by PCR (Weir et al. 2012). Gene sequences were deposited in GenBank (GenBank accession no. MW398863 for ACT, MW886232 for CHS-1, MW398864 for GAPDH, MW398865 for ITS). BLAST analysis revealed that DNA sequences of YX2-5-2 at the ITS, GAPDH, ACT, and CHS-1 loci showed 100%, 99.25%, 100%, and 99.33% sequence identity, respectively to their corresponding loci in strains ZH6 (GenBank accession no. MT476840.1), ICKP18B4 (LC494274.1), YN17 (MN525804.1), and ICKG4 (LC469131.1) of C. fructicola. A Maximum Likelihood phylogenetic tree based on the combined ACT, CHS-1, ITS and GAPDH sequences revealed that the representative isolate YX2-5-2 clustered with C. fructicola. In addition, the morphological features of YX2-5-2 were similar to C. fructicola which has been reported (Weir et al. 2012). Pathogenicity was tested using isolate YX2-5-2 by inoculating leaves of 2-year-old C. yuhsienensis. Four leaves of each healthy C. yuhsienensis were sprayed with a conidial suspension (105 conidial/mL) of isolate YX2-5-2, and the above steps were repeated three times. Two additional mock-inoculated control plants were sprayed with sterilized liquid potato dextrose medium. The plants were incubated in a greenhouse at 28℃ and 90% humidity with a 12 h photoperiod. Anthracnose-like symptoms were observed 5 days post-inoculation. The control plant tissues remained healthy. C. fructicola was re-isolated on PDA from lesions, and the morphological features were consistent with YX2-5-2, confirming Koch's postulates. To our knowledge, this is the first report of anthracnose of C. yuhsienensis caused by C. fructicola in China. Anthracnose of Camellia. oleifera has been reported for a long time (H. Li et al. 2016). C. yuhsienensis, as a wild relative of C. oleifera (commonly known as tea-oil tree), has been concerned about its resistance to anthracnose. Therefore, the occurrence of C. yuhsienensis anthracnose hindered the control of anthracnose tea-oil tree. This finding will lay the foundation for studying the pathogenesis of anthracnose of tea-oil tree and developing effective prevention methods.

First report of Phaeoacremonium oleae and P. viticola associated with olive trunk diseases in Italy.

Over 300 trunk, branch and stem samples with vascular discolouration, necrotic wood and shoot death were collected from olive (Olea europaea) orchards in Lecce, Brindisi, Bari and Foggia provinces (Apulia region, Italy) from October to May from 2013 to 2019. Small chips of symptomatic wood samples were surface sterilised (5% NaOCl, 3 min; 70% ethanol, 30 s), rinsed (sterile distilled water, ×3), and placed onto potato dextrose agar (PDA) plates amended with 500 ppm streptomycin sulphate. After 14 days at 25 °C in the dark, hyphal tips of growing fungi, including different taxa, for instance Phaeoacremonium and Botryosphaeriaceae spp., were transferred to new PDA plates and incubated until sporulation. Monoclonal colonies resembling Phaeoacremonium-like genus (Mostert et al. 2006) were selected for further study, and genomic DNA of 59 representative isolates was extracted (Carlucci et al. 2013). Partial actin and β-tubulin genes were amplified with primers ACT-513F/ACT-783R (Carbone & Kohn 1999), and T1(O'Donnell & Cigelnik 1997) and Bt2b (Glass & Donaldson 1995), respectively. The sequenced amplicons were compared by BLAST algorithms with reference strains of Phaeoacremonium spp. retrieved from GenBank. Forty-four isolates showed 99% to 100% similarity with reference strains P. italicum, P. minimum, P. parasiticum, P. scolyti and P. sicilianum (Carlucci et al. 2015), nine with P. oleae, and six with P. viticola. Actin and β-tubulin sequences of P. oleae (Pm14) and P. viticola (Pm34) were submitted to GenBank (MW714561, MW714563; MZ318697, MZ318696). Microscopy of P. oleae isolates showed: conidiophores branched and unbranched, (18.7-)21.9-57.1(-67.8) × (2.9-)3.3-4.7(-5.2) (mean, 38.9×4.1) μm (n=30); conidia oblong-ellipsoidal to obovoid or subcylindrical 3.4 to 5.5 μm long, and 1.5 to 2.4 (mean, 4.6 × 2.2) μm wide (n=30). Microscopy of P. viticola isolates showed: conidiophores subcylindrical, branched at base (6.7-)8.9-27.2(-29.3) × (2.0-)2.6-3.3(-3.7) (mean, 21.4 × 3.2) μm (n=30); conidia oblong-ellipsoidal to obovoid or subcylindrical 3.3 to 6.8 μm long, and 1.1 to 2.2 (mean, 4.2 × 1.6) μm wide (n=30). In spring 2020, artificial inoculations were carried out with P. oleae (Pm14, Pm46) and P. viticola (Pm34, Pm43) strains on 10 healthy, 2-year-old olive seedlings cultivar 'Coratina'. Agar plugs (diameter, 0.3-0.5 cm) from 10-day-old cultures grown on water agar at 23 (±2) °C were inserted under the bark of small wounds in the stems (length, 0.4-1.0 cm) made with a sterile scalpel. After inoculation, the wounds were wrapped with wet sterile cotton wool and sealed with Parafilm. Ten control olive seedlings were inoculated with sterile agar plugs. The experiment was replicated three times. All inoculated young olive plants were grown in pots in a greenhouse without temperature control. After 120 days, inoculated plants showed decline symptoms, and when cut longitudinally, brown streaks were observed in the wood. For P. oleae these streaks measured 3.0-5.5 cm long (standard deviation [SD], 0.9 cm, and for P. viticola they were 1.8-3.5 cm (SD, 0.62). Both fungal species were re-isolated from the symptomatic wood from 85% and 80%, respectively, of these inoculated olive seedlings, fulfilling Koch's postulates. No symptoms were observed from olive seedlings used as control. P. oleae was first described as a fungal pathogen of wild olive (Olea europaea subsp. cuspidate) in South Africa by Spies et al. (2018), and P. viticola as a fungal pathogen of grapevine in France by Dupont et al. (2000). To the best of our knowledge, this is the first report of P. oleae associated with olive trunk disease in Italy, and the first report of P. viticola associated with olive trunk disease worldwide.

First report of Colletotrichum fructicola causing anthracnose on cherry (Prunus avium) in China.

Cherry (Prunus avium) has become an important economical fruit in China. In October 2020, a leaf spot disease was found on cherry in the orchard of Taizhou Academy of Agriculture Sciences, Zhejiang, China. The symptoms appeared as small, water-soaked spots on the leaves, which later became larger, dark brown, and necrotic lesions of 1 cm to 3 cm in width, 4 cm to 8 cm in length. Disease incidences of approximately 60% of the leaves were observed by sampling five locations. To isolate the causing agent, small fragments from five target symptomatic leaves were surface-sterilized with 1.0% sodium hypochlorite solution for 1 min and then rinsed three times with sterilized water. Afterwards the leaf fragments were air-dried, plated onto potato dextrose agar (PDA) medium, and incubated at 25 ℃ in the dark for 2 days. The pure cultures were obtained by transferring hyphal plug of 2 mm in diameter onto PDA, which followed single spore isolation. The colony morphology showed light to dark gray, cottony mycelium, with the underside of the culture became brownish after 7 days. Conidia (n = 28) were hyaline, smooth-walled, cylindrical, aseptate, broadly rounded ends, and average size around 3.84 × 12.82 μm (2.99 to 4.87 × 10.27 to 15.68 μm). Appressoria (n = 27) were mostly brown, ovoid and slightly irregular in shape, and average size around 8.04 × 9.68 μm (6.29 to 9.67 × 9.32 to 12.06 μm). Perithecia average size is 106.25 μm, textura angularis, thick-walled. Asci 26.35-49.18 × 5.00-12.03 μm (average size 37.44 × 7.80 μm, n = 17), unitunicate, thin-walled, clavate or cymbiform. Ascospores 13.69-20.93 × 3.86-6.69 μm (average size 16.00 × 5.42 μm, n = 30), one-celled, hyaline, one or two large guttulate at the centre, slightly rounded ends. The morphological characteristics matched well with previous descriptions of Colletotrichum species of C. gloeosporioides species complex, including C. fructicola (Prihastuti et al. 2009; Fu et al. 2019). The identity of two representative isolates (cf2-3 and cf4-4) from different leaves was confirmed by means of multi-locus gene sequencing. To this end, genomic DNA was extracted by the Plant Direct PCR kit (Vazyme Biotech Co., Ltd, China). Molecular identification was conducted by sequencing the internal transcribed spacer (ITS) rDNA region, partial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, partial actin (ACT) gene, partial beta-tubulin 2 gene (TUB2), and partial chitin synthase gene (CHS). The obtained sequences have been deposited in GenBank under accession numbers MW581851 and MW581852 (ITS), MW590586 and MW590587 (GAPDH), MW616561 and MW616562 (ACT), MW729380 and MW729381 (TUB2), MW729378 and MW729379 (CHS). The results of Basic Local Alignment Search Tool (BLAST) analysis revealed that the ITS, GAPDH, ACT, TUB2 and CHS sequences of both isolates matched with 100% identity to Colletotrichum fructicola culture collection sequences in GenBank database (JX010165, JX009998, JX009491, JX010405, and JX009866 respectively). These morphological characteristics and molecular analyses allowed the identification of the pathogen as C. fructicola. Koch's postulates were performed with healthy detached cherry leaves of cultivar namely 'HongMi' from Taizhou Academy of Agriculture Sciences. Surface-sterilized leaves were inoculated with five-day-old cultures of C. fructicola mycelial discs of 2 mm in diameter after being wounded with a needle or non-wounded. Control leaves were inoculated with discs of same size PDA agar. Treated leaves were incubated at 25 ℃ in the dark at high relative humidity. Anthracnose symptoms appeared within 3 days both on non-wounded and wounded inoculation approaches. Mock-inoculated controls remained asymptomatic. Biological repetitions were carried out three times. The fungus was reisolated from infected leaves and confirmed as C. fructicola following the methods described above. Until recently, it has been found that C. fructicola can infect tea, apple, pear, Pouteria campechiana in China (Fu et al. 2014; Li et al. 2013; Shi et al. 2018; Yang et al. 2020). To the best of our knowledge, this is the first report of C. fructicola on cherry in China.

First report of Phaeoacremonium amygdalinum associated with almond dieback and wood disease in Italy.

In the last 10 years, almond (Prunus dulcis (Mill.) Webb) cultivation areas in the region of Apulia (southern Italy) have increased. A recent survey in young almond orchards recorded characteristic disease symptoms including rapid collapse of branches, chlorosis of leaves (with sudden wilting and death) in June/July, and bud and shoot dieback in December-March. Internal wood symptoms on stems consisted of brown vascular streaking that led to death of young apical shoots within a few weeks. Wood samples were collected and taken to the laboratory, where they underwent disinfection according to Fisher et al. (2002). Fungal isolation techniques were applied, with small pieces of wood tissue (0.5-1.0 cm) placed on potato dextrose agar (PDA; Oxoid Ltd.) supplemented with 300 mg/L streptomycin sulphate, and incubated at 23 ±2 °C in the dark. After 10 days, based on morphological features carried out by microscopic studies, 85% (n = 55) of the resulting fungal colonies (N = 65) were preliminarily attributed to known fungal pathogens of woody tissues, of which 12.4% (n = 8) belonged to the Phaeoacremonium genus. These last were purified by transferring single germinating conidia onto fresh PDA plates. In particular, the conidiophores were mostly short, usually unbranched, erect to flexuous, up to 5-septate, brown to pale brown, verruculose on the lower part, (12-)15.7-41(-55) (av. -29) μm long and 1.6-3.2 (av. 2.1) μm wide (n = 30). Conidia hyaline, oblong ellipsoidal-obovoid or subcylindrical (3-)4-5 μm long and 1.5-3 (av. = 4.4 × 2.1) μm wide (n =30). All Phaeoacremonium isolates were successfully subjected to genomic DNA extraction, according to Carlucci et al. (2013). Partial actin and β-tubulin genes were amplified with the ACT-513F/ACT-783R (Carbone and Kohn 1999) and T1 (O'Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995) primers, respectively. Consensus sequences were compared with reference strains (extype) in the GenBank database, using the Basic Local Alignment Search Tool (BLAST). In particular, all isolates showed 99-100% similarity with reference strains of Phaeoacremonium amygdalinum Gramaje, Armengol & L. Mostert (JN191301; JN191305). Actin and β-tubulin sequences of P. amygdalinum strain Pm10 were deposited in GenBank (MW716265; MW714562). In spring 2020, artificial inoculations with two isolates of P. amygdalinum (Pm10; Pm15) were carried out on 10 healthy, 2-year-old almond seedlings of cv. 'Filippo Cea'. Agar plugs (diameter, 0.3-0.5 cm) taken from 10-day-old cultures grown on PDA at 23 ±2 °C were inserted into small wounds under the bark of the stems (length, 0.4-1.0 cm) made with a sterile scalpel. After inoculation, the wounds were wrapped with wet sterile cotton wool and sealed with Parafilm. Ten almond seedling were used as controls, being inoculated with sterile agar plugs. The experiment was replicated three times. The inoculated young almond plants were grown in pots in a greenhouse without temperature control. After 150 days, the inoculated plants showed decline symptoms and internal longitudinal brown wood streaking (average, length, 2.7-6.4 cm). Phaeoacremonium amygdalinum was reisolated from symptomatic wood of 95% of the experimental almond seedlings, thus fulfilling Koch's postulates. No symptoms were observed on almond seedling used as controls. Phaeoacremonium amygdalinum was first described as a fungal pathogen of almond in Spain (Gramaje et al., 2012). To the best of our knowledge, this is the first report of P. amygdalinum associated with almond dieback disease in Italy. References Carbone I. and Kohn L.M. 1999. Mycologia 91:553. Carlucci A. et al. 2013. Phytopathol. Mediterr. 52:517. Fisher et al 2002. Mycol. Prog. 1:315 Glass N. L. and Donaldson G. C. 1995. J. Cl. Microbiol. 41:1332. Gramaje et al. 2012. Persoonia 28:1. O'Donnell K. and Cigelnik E. 1997. Mol. Phylogenetics Evol 7:103.

First Report of a Leaf Blight Caused by Pyricularia pennisetigena on Cenchrus echinatus in Paraguay.

The genus Pyricularia contains several fungal species known to cause diseases on plants in the Poaceae family (Klaubauf et al. 2014; Wang et al. 2019). While sampling for P. oryzae during March-2015 and April-2018, common weed Cenchrus echinatus L. was observed with leaf lesions in and around experimental wheat fields in the departments of Canindeyú and Itapúa. C. echinatus samples from both locations displayed similar leaf lesions, varying from small light brown pinpoint to elliptical brown lesions with greyish center. Symptomatic leaves were surface disinfested and cultured on potato dextrose agar (PDA) amended with 1% gentamicin at 25°C. Two monosporic isolates were obtained, one from Itapúa (ITCeh117) and the other from Canindeyú (YCeh55). The isolates were subsequently grown on oatmeal agar (OA) and PDA under a 12-h photoperiod at 25°C and evaluated after ten days for colony diameter, sporulation, macroscopic and microscopic features. Colonies on OA reached up to 4.8 cm diameter and were light grey, whereas colonies on PDA reached up to 5.3 cm diameter and were brown with grey centers, with cottony mycelium and broad white rims. Mycelium consisted of smooth, hyaline, branched, septate hyphae 4-4.5 µm diameter. Conidiophores were erect, straight or curved, unbranched, medium brown and smooth. Conidia were solitary, pyriform, pale brown, smooth, granular, 2-septate, 32-33 × 9-10 μm; truncated with protruding hilum and varied in length from 1.0 to 1.5 μm and diameters from 2.0 to 2.2 μm. Both isolates were similar and identified as Pyricularia pennisetigena, according to morphological and morphometric characteristics (Klaubauf et al. 2014). Subsequently, genomic DNA was extracted from each isolate using the primers described in Klaubauf et al. (2014) to amplify and sequence the internal transcribed spacers (ITS), partial large subunit (LSU), partial RNA polymerase II large subunit gene (RPB1), partial actin gene (ACT), and partial calmodulin gene (CAL). Sequences from each isolate (YCeh55/ITCeh117) were deposited in GenBank with the following submission ID for ITS: MN947521/MN947526, RPB1: MN984710/MN984715, LSU: MN944829/MN944834, ACT: MN917177/MN917182, and CAL: MN984688/MN984693. Phylogenetic analysis was conducted using the software Beast v1.10.4. The results obtained from the concatenated matrix of the five loci placed these isolates in the P. pennisetigena clade. To confirm pathogenicity, each isolate was adjusted to 5×104 conidia/ml of sterile water and C. echinatus plants were sprayed with the conidial suspension for isolate YCeh55, ITCeh117 or sterile water using an oilless airbrush sprayer until runoff. The three treatments were kept in the greenhouse at 25-28°C and about 75% relative humidity under natural daylight. Each treatment included three to five inoculated plants and 10 leaves were evaluated per treatment. Symptoms were observed 8-15 days after inoculation and were similar to those originally observed in the field for both isolates, whereas the control plants remained asymptomatic. P. pennisetigena was re-isolated from the inoculated leaves fulfilling Koch's postulates. To our knowledge, this is the first report of leaf blight on C. echinatus caused by P. pennisetigena in Paraguay. The occurrence of P. pennisetigena in the region and its ability to infect economically important crops such as wheat and barley (Klaubauf et al. 2014; Reges et al., 2016, 2018) pose a potential threat to agriculture in Paraguay.

First Report of Anthracnose of Papaya (Carica papaya L.) Caused by Colletotrichum siamense in China.

Papaya (Carica papaya L.) is a rosaceous plant widely grown in China, which is economically important. Anthracnose caused by Colletotrichum sp. is an important postharvest disease, which severely affects the quality of papaya fruits (Liu et al., 2019). During April 2020, some mature papaya fruits with typical anthracnose symptoms were observed in Fusui, Nanning, Guangxi, China with an average of 30% disease incidence (DI) and over 60% DI in some orchards. Initial symptoms of these papayas appeared as watery lesions, which turned dark brown, sunken, with a conidial mass appearing on the lesions under humid and warm conditions. The disease severity varied among fruits, with some showing tiny light brown spots, and some ripe fruits presenting brownish, rounded, necrotic and depressed lesions over part of their surface. Samples from two papaya plantations(107.54°E, 22.38°N) were collected, and brought to the laboratory. Symptomatic diseased tissues were cut into 5 × 5 mm pieces, surface sterilized with 2% (v/v) sodium hypochlorite for 1 minute, and rinsed three times with sterilized water. The pieces were then placed on potato dextrose agar (PDA). After incubation at 25°C in the dark for one week, colonies with uniform morphology were obtained. The aerial mycelium on PDA was white on top side, and concentric rings of salmon acervuli on the underside. A gelatinous layer of spores was observed on part of PDA plates after 7 days at 28°C. The conidia were elliptical, aseptate and hyaline (Zhang et al., 2020). The length and width of 60 conidia were measured for each of the two representative isolates, MG2-1 and MG3-1, and these averaged 13.10 × 5.11 μm and 14.45 × 5.95 μm. DNA was extracted from mycelia of these two isolates with the DNA secure Plant Kit (TIANGEN, Biotech, China). The internal transcribed spacer (ITS), partial actin (ACT), calmodulin (CAL), chitin synthase (CHS), β-tubulin 2 (TUB2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) regions were amplified by PCR and sequenced. The sequences were deposited into GenBank with accessions MT904003, MT904004, and MT898650 to MT898659. BLASTN analyses against the GenBank database showed that they all had over 99% identity to the type strain of Colletotrichum siamense isolate ICMP 18642 (GenBank accession numbers JX010278, GQ856775, JX009709, GQ856730, JX010410, JX010019) (Weir et al., 2012). A phylogenetic tree based on the combined ITS, ACT, CAL, CHS, TUB2 and GAPDH sequences using the Neighbor-joining algorithm also showed that the isolates were C. siamense. Pathogenicity tests were conducted on 24 mature, healthy and surface-sterilized papaya fruits. On 12 papaya fruits, three well separated wounded sites were made for inoculation, and for each wounded site, six adjacent pinhole wounds were made in a 5-mm-diameter circular area using a sterilized needle. A 10 µl aliquot of 1 × 106 conidia/ml suspension of each of the isolates (MG2-1 and MG3-1) was inoculated into each wound. For each isolate, there were six replicate fruits. The control fruits were inoculated with sterile distilled water. The same inoculation was applied to 12 non-wound papaya fruits. Fruits were then placed in boxes which were first washed with 75% alcohol and lined with autoclaved filter paper moistened with sterilized distilled water to maintain high humidity. The boxes were then sealed and incubated at 28°C. After 10 days, all the inoculated fruits showed symptoms, while the fruits that were mock inoculated were without symptoms. Koch's postulates were fulfilled by re-isolation of C. siamense from diseased fruits. To our knowledge, this is the first report of C. siamense causing anthracnose of papaya in China. This finding will enable better control of anthracnose disease caused by C. siamense on papaya.

First Report of Leaf Spot Caused by Colletotrichum fructicola and C. siamense on Zizyphus mauritiana in Guangxi, China.

Zizyphus mauritiana Lam. is an important tropical fruit tree and has significant economic value. It is widely planted in Hainan, Guangdong, Guangxi and Fujian provinces in China (Yang et al. 2017). In March 2019, leaf spot was observed on leaves of Z. mauritiana at Bagui fields in Nanning, Guangxi, China, with incidence exceeding 50%. Symptomatic leaves developed a yellow to tan-brown sunken lesion and finally abscised. To isolate the pathogen causing the symptoms, small pieces (5 × 5 mm) of infected leaves were surface sterilized by exposure to 75% ethanol for 10 sec, 1% sodium hypochlorite for 1 min and rinsed three times in sterile water. Fifty pieces were isolated, surface sterilized, and pieces were plated onto potato dextrose agar (PDA) and grown at 28°C for 7 days. The isolation rate of Colletotrichum species was 100%. Three representative isolates (DQZ3-1, DQZ3-2 and DQZ3-3) were selected for further study. Mycelia were greyish-white for all three isolates, with isolate DQZ3-1 also appearing dark green in the center of the colony. Conidia were elliptical, aseptate and hyaline, with sizes of 13.4 ± 0.12 µm × 5.7 ± 0.1 µm, 14.8 ± 0.1 µm × 5.8 ± 0.1 µm and 15.1 ± 0.1 µm × 5.5 ± 0.1 µm for DQZ3-1, DQZ3-2 and DQZ3-3, respectively. Genomic DNA was extracted using the DNAsecure Plant Kit [Tiangen Biotech (Beijing) Co., Ltd] and the internal transcribed spacer (ITS), partial actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), beta-tubulin (TUB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were sequenced (Weir et al. 2012). Phylogenetic analysis of the three isolates was performed with MEGA-X (Version 10.0) based on sequences of multiple loci (ITS, ACT, CAL, CHS-1, TUB2 and GAPDH) using Maximum Likelihood analysis. Isolate DQZ3-1 was identified as C. fructicola, and the other two isolates, DQZ3-2 and DQZ3-3, were identified as C. siamense (accessions MT039396 to MT039410, for ACT, CAL, CHS-1, GAPDH and TUB2 of DQZ3-1, DQZ3-2 and DQZ3-3; MT041651 to MT041653 for ITS of DQZ3-1, DQZ3-2 and DQZ3-3). Pathogenicity tests were conducted on 1-year-old plants. Young, healthy leaves were artificially wounded by gently scratching with a sterile needle and 10 µl droplets of conidial suspension (106 spores/ml) applied per wound site for each isolate. Some wounded leaves were inoculated with 10 µl droplets of water as controls. Each isolate was inoculated onto three plants, with 15 leaves at least for each plant, same as controls. All inoculated plants were sprayed with water and covered with plastic bags to maintain high humidity. Symptomatic lesions were observed on the inoculated leaves after 7 days at 28°C, whereas no symptoms were observed on the control leaves. To fulfill Koch's postulates, fungi were re-isolated from 50 symptomatic leaf pieces and fungi re-isolated from each leaf piece were morphologically identical to the inoculated isolates, for a 100% isolation frequency. To our knowledge, this is the first report of leaf spot caused by C. fructicola and C. siamense on Z. mauritiana worldwide. This research may accelerate the development of future epidemiological studies and management strategies for anthracnose caused by C. fructicola and C. siamense on Z. mauritiana.

First record of Colletotrichum alienum Causing postharvest Anthracnose disease of mango fruit in China.

Mango (Mangifera indica L.) is one of the world's most significant economic fruit crops, and China is the second-largest producer of mango (Kuhn et al., 2017). Postharvest mango anthracnose is caused by Colletotrichum species and reduce the self-life of mature fruit (Wu et al., 2020). Colletotrichum species also cause postharvest anthracnose and fruit rot disease of Apple, Banana and Avocado (Khodadadi et al., 2020; Vieira et al., 2017; Sharma et al., 2017). In July 2019, mango fruits cv. 'Jin-Hwang' were observed at different fruit markets (39°48'42.1"N 116°20'17.0"E) of the Fengtai district, Beijing, China, exhibiting typical symptoms of anthracnose including brown to black lesions in different size (≤ 2 cm) with identified border on the mango fruit surface. Later, the lesions were coalesced and extensively cover the surface area of the fruit. The lesions were also restricted to peel the fruit and pathogen invaded in the fruit pulp. About 30% of mango fruits were affected by anthracnose disease. The margins of lesions from infected mango fruits (n=56) were cut into 2 × 2 mm pieces, surface disinfected with NaClO (2% v/v) for 30 s, rinsed thrice with distilled water for 60s. These pieces were placed on PDA medium and incubated at 25°C for 7 days. Pure culture of fungal isolates was obtained by single spore isolation technique. Initially, the fungal colony was off white, and colony extended with time, turning light gray at the center. The morphological examination revealed that conidia were hyaline, oblong, and unicellular. The conidia were measured from 10 days old culture and dimensions varied from 13.3 to 15.8 µm in length and 4.6 to 6.1 µm in width. For molecular identification, a multi-locus sequence analysis; the Internal Transcribed Spacers (ITS) region, partial actin (ACT) gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and chitin synthase (CHS-1) gene were amplified by using the primer sets ITS1/4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), GDF1/GDR1 (Guerber et al. 2003) and CHS1-79F/CHS-1-354R (Carbone and Kohn 1999) respectively. The partial sequences of MTY21 were deposited to GenBank accessions (MT921666 (ITS), MT936119 (ACT), MT936120 (GAPDH) and MT936118 (CHS-1). All obtained sequences showed 100% similarity with reported sequences of Colletotrichum alienum ICMP.18691 with accessions numbers JX010217 (ITS), JX009580 (ACT), JX010018 (GAPDH) and JX009754 (CHS-1) which represented the isolate MTY21 identified as C. alienum by constructing Maximum Likelihood phylogenetic tree analysis using Mega X (Kumar et al., 2018). For the confirmation of Koch's postulates, the pathogenicity test was conducted on 36 fresh healthy mango fruits for each treatment. Fruits were punctured with the help of a sterilized needle to create 2mm2 wounds and inoculated with 10µL inoculum (107 spores/mL) of MTY21. Control mango fruits were inoculated with 10µL sterilized distilled water and incubated at 25 °C with 90% relative humidity. The lesions appeared at the point of inoculation and gradually spread on the fruit surface after 7 days post inoculation. The symptoms were similar to the symptoms on original fruit specimens. The re-isolated fungus was identified as C. alienum based on morphological and molecular analysis. Mango anthracnose disease caused by several Colletotrichum species has been reported previously on mango in China (Li et al., 2019). Liu et al. (2020) reported C. alienum as the causal organism of anthracnose disease on Aquilaria sinensis in China. C. alienum has been previously reported causing mango anthracnose disease in Mexico (Tovar-Pedraza et al., 2020) To our knowledge, this is the first report of C. alienum causing postharvest anthracnose of mango in China. The prevalence of C. alienum was 30% on mango fruit which reflects the importance of this pathogen as a potential problem of mango fruit in China.

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

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

Unveiling the Hidden Diversity of Rock-Inhabiting Fungi: Chaetothyriales from China.

Rock-inhabiting fungi (RIF) are nonlichenized fungi that naturally colonize rock surfaces and subsurfaces. The extremely slow growth rate and lack of distinguishing morphological characteristics of RIF resulted in a poor understanding on their biodiversity. In this study, we surveyed RIF colonizing historical stone monuments and natural rock formations from throughout China. Among over 1000 isolates, after preliminary delimitation using the internal transcribed spacer region (ITS) sequences, representative isolates belonging to Trichomeriaceae and Herpotrichiellaceae were selected for a combined analysis of ITS and the nuclear ribosomal large subunit (nucLSU) to determine the generic placements. Eight clades representing seven known genera and one new genus herein named as Anthracina were placed in Trichomeriaceae. While, for Herpotrichiellaceae, two clades corresponded to two genera: Cladophialophora and Exophiala. Fine-scale phylogenetic analyses using combined sequences of the partial actin gene (ACT), ITS, mitochondrial small subunit ribosomal DNA (mtSSU), nucLSU, the largest subunit of RNA polymerase II (RPB1), small subunit of nuclear ribosomal RNA gene (SSU), translation elongation factor (TEF), and β-tubulin gene (TUB) revealed that these strains represented 11 and 6 new species, respectively, in Trichomeriaceae and Herpotrichiellaceae. The 17 new species were described, illustrated for their morphologies and compared with similar taxa. Our study demonstrated that the diversity of RIF is surprisingly high and still poorly understood. In addition, a rapid strategy for classifying RIF was proposed to determine the generic and familial placements through preliminary ITS and nucLSU analyses, followed by combined analyses of five loci selected from ACT, ITS, mtSSU, nucLSU, RPB1, and/or the second subunit of RNA polymerase II gene (RPB2), SSU, TEF, and TUB regions to classify RIF to the species level.

First report of Leaf Spot Associated with Boeremia exigua on White Clover in China.

White clover (Trifolium repens L.) is an important perennial legume forage widely cultivated in China (Zhang et al. 2016). In April 2018, severe necrotic lesions on leaves were observed in a cultivated white clover field in Chongqing, China. Approximately 90% of plants in the field were affected. Leaf spots were amphigenous, dark-brown, elliptic to subcircular, with diameter ranging between 1 to 12 mm, well defined by brown margins and yellow halos. Severely infected leaves became withered and abscised. Stems and flowers were not affected by the disease. Symptomatic leaves were 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 from margins of individual lesions were placed on potato dextrose agar (PDA) amended with 50 mg/L of chloramphenicol and incubated at 25℃ in the dark. An olivaceous gray fungal colony was consistently isolated (90.5% isolation frequency). After 15 days of incubation, subglobose, black pycnidia developed in the cultural medium. Conidia were hyaline, ellipsoid to oblong, nonseptate(n = 50), ranging from 4.0 to 7.5 μm long (5.6 ± 2.3µm) × 2.0 to 3.8 μm wide(2.8 ± 1.0 µm). On the basis of its morphological characteristics, the fungus was identified as Boeremia sp. (Aveskamp et al. 2010). To confirm the identity, the internal transcribed spacer region (ITS), large subunit ribosomal RNA (LSU), partial actin (ACT), RNA polymerase II second largest subunit (rpb2) and beta-tubulin (tub2) genes were amplified with primers ITS1/ITS4, LR0R/LR7, ACT-512F/ACT-783R, RPB2-5F2/fRPB2-7cR, and Btub2Fd/Btub4Rd, respectively, in eight representative isolates and sequenced(Aveskamp et al. 2009; Chen et al. 2015). BLAST results showed 100% identity of the ITS (506/506 nucleotides), LSU (966/966 nucleotides), ACT (244/244 nucleotides) and tub2 (297/297 nucleotides) sequences with those of B. exigua (KY419536, MK398746, EU880878, and MK514090) and 99.83% identity of those of the rpb2 (593/594 nucleotides) sequence with B. exigua (KT389572).Based on morphology and DNA sequence analysis, the associated fungus was identified as B. exigua. Representative sequences of one isolate (BT2-1) were deposited in GenBank (MN826339, MN836592, MT265217, MT265218 and MT265219). In a pathogenicity test, ten 2-month-old potted white clover plants were spray-inoculated with a spore and mycelial suspension (approximately 105 CFU/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 3 days to maintain high humidity. After 10 days, typical dark-brown lesions similar to those seen in naturally infected leaves developed on the inoculated leaves and not on the control plants. B. exigua was reisolated from the lesions, thus completing Koch's postulates. There is some evidence that B exigua is capable of invading seedling root tissue of white clover and causing necrotic lesions on roots (Skipp and Christensen,1982). However, to our knowledge, this is the first report of leaf spot on T. repens caused by B. exigua. This disease severely reduces forage quality and yield. Proper identification of the causal organism is essential in formulating management strategies.

First Report of Leaf Blight Caused by Cladosporium perangustum on Livistona chinensis in China.

Livistona chinensis (Jacq.) R.Br. ex Mart. belonging to monocotyledonous Palmaceae, is widely distributed in Eastern Asia, and a common ornamental plant in Southern China (Li et al. 2019, Wu et al. 2019). In November 2019, epidemics of leaf blight were discovered on aged leaves of L. chinensis in the campus of Sichuan Agricultural University, where disease incidence was up to 90% among the cultivated plants, but without resulting in the death of the host. Initial symptoms appeared chlorotic spots on the upper part of leaves, then spots expanded and turned brown. Subsequently, the enlarged spots developed necrotic tissues in the center with brown to dark brown margin. At later stages, conidia and conidiophores were observed on the surface of lesions with the aid of a microscope. As the disease progressed, multiple lesions usually coalesced to cause extensive tissue necrosis at third or more parts of the leaf. Three isolates were obtained from diseased leaves with a single spore isolation referred to Chomnunti et al. (2014), and cultured on potato dextrose agar (PDA) under a 12-h photoperiod and 25±1℃. The colonies were gray to gray-olivaceous with an olivaceous-black reverse side, and measured approximately 7 cm in diameter after 6 days. The conidiophores were macronematous, semimacronematous or micronematous, erect, straight or slightly flexuous, filiform to narrowly cylindrical-oblong, occasionally branched, pale olivaceous-brown, wider at the base, 1 to 8 septate, 14-104 × 3-6.8 µm (x̅ =57.7 × 4.9 µm, n=30). Conidiogenous cells were integrated, terminal, intercalary or pleurogenous, cylindrical-oblong, 9-21 × 4-5.9 µm (x̅ =15 × 4.9 µm, n=20), thick walled, usually aseptate, occasionally 1-2-septate, with 1-5 apically crowed loci. Ramoconidia were almost oblong, aseptate, 4-8.4 × 3-5.7 µm (x̅ =6.9 × 4.6 µm, n=20). Conidia were numerous, catenate, in branched chains, small terminal conidia, subglobose or ovoid, and intercalary conidia were ovoid, limoniform, fusiform, aseptate, 2.9-5.5 × 2-5 µm (x̅ =4 × 3.5 µm, n=20). The representative isolate SICAUCC 20-0007 was used for Genomic DNA extraction, and the internal transcribed spacer (ITS), translation elongation factor 1 alpha (TEF 1-α) and partial actin (ACT) regions were amplified using the primer pairs ITS5/ITS4 (White et al. 1990), EF1-728F/EF-2 (O'Donnell et al. 1998, Carbone et al. 1999) and ACT-512F/ACT-783R (Carbone et al. 1999), respectively. Nucleotide BLAST in GenBank revealed identity with those of C. perangustum, the ITS (MT427730) had 100% identity with MF473185 and MF473177, TEF 1-α (MT441922) had 100% identity with HM148379 and MF473595, ACT (MT441917) had 100% identity with HK521577 and MH047335. The phylogenetic tree combined with ACT, ITS, and TEF 1-α genes (Fig. 1) and morphological characteristics confirmed the identification as C. perangustum defined by Bensch et al. (2010). To determine pathogenicity, five potted plants were inoculated with conidial suspensions (105 conidia/ml) prepared from 1-week-old cultures of the isolate SICAUCC 20-0007 onto the wounded sites via pin-prick inoculation (five to eight leaves per plant with roughly 1 to 2-year-old), and the same amount of healthy plant was sprayed with distilled water as controls. Plants were sprayed regularly with distilled water every day and placed under a 12-h photoperiod and 25±1℃. About fifteen days later, faint yellow to yellowish-brown spots were found on inoculated leaves and were similar to those previously observed and no symptoms developed on the control plants. After a month, leaf blight was observed and the pathogenic fungus was re-isolated from the inoculated tissues. Previous reports have shown that C. perangustum causes leaf spot on Myrica rubra (Lour.) Siebold & Zucc. in China (Lu et al. 2015). To our knowledge, this is the first report of C. perangustum causing leaf blight of L. chinensis in the world. This disease potentially reduces the ornamental value under favorable conditions, and proper control strategies should be implemented.

First Report of Didymella americana on Lima Bean (Phaseolus lunatus) in Delaware and Maryland

Delaware and Maryland have a long history of commercial production of lima bean (Phaseolus lunatus) for the processing market. In 2015, we observed lima bean plants that had irregular tan lesions with reddish borders in two fields in Sussex County, DE. Lesions ranged from 0.4 to 2.0 cm in diameter. Approximately 10% of leaves were symptomatic, and severity ranged between 5 and 10%. Symptomatic leaves (n = 6 to 10) were collected in September and October from each field for isolation and evaluation. Similar symptoms were observed on lima bean plants at the University of Maryland Lower Eastern Shore Research and Education Center in 2018, and additional symptomatic leaves (n = 6) were collected. Isolations were conducted from six diseased leaves per field (n = 18) by placing 2- to 4-mm sections of leaf on potato dextrose agar (PDA). All lesions resulted in isolates that appeared similar in morphology. Hyphae from the representative colonies were placed on water agar, and after 24 h, hyphal tips were excised. The hyphal tip procedure was repeated once to obtain pure cultures. Six isolates from Delaware and one from Maryland were stored at –80°C until use. Morphological evaluation of isolates grown on oat agar (OA), malt extract agar (MEA), and PDA revealed conidial morphology and dimensions consistent with previously published reports of Didymella americana (Aveskamp et al. 2010; Gorny et al. 2016). Two isolates, De15-002 and Md18-06, were selected for molecular analysis. DNA was extracted from colonies grown on OA; the internal transcribed spacer (ITS) region was amplified with primers ITS1/ITS4, and the partial actin locus region was amplified with primers act-512/act-783. Basic local alignment searches compared sequences to the ITS (GenBank accession no. FJ426972) and partial actin (FJ426870) sequences of D. americana reference strain CBS 185.85 (Chen et al. 2017; Gorny et al. 2016). The ITS sequences of isolates De15-002 and Md18-06 (MN698269 and MN944417, respectively) shared a 100% similarity with the reference strain. The partial actin sequences of isolates De15-002 (MN820723) and Md18-06 (MN820722) were also identical and differed from the reference sequence by 1 bp (99% similarity). To fulfill Koch’s postulates, four 3-week-old lima bean plants (cv. C-Elite) were sprayed with a conidial suspension (3 × 10⁵ conidia/ml) from 30-day-old cultures grown on MEA or with distilled water (control). Following 28 days of incubation at approximately 20°C, lesions were observed on all plants that were inoculated with isolates De15-001, De15-002, De15-004, De15-005, De15-007, De15-009, and Md18-06, but not on the water-inoculated control plants. Reisolations (n = 4 per isolate) onto water agar and MEA were examined after 7 days to confirm morphology similar to the original isolates. Based on the morphological and molecular characteristics, the pathogen was identified as D. americana. This is the first report of D. americana on lima bean in the Mid-Atlantic region of the United States. Lima bean is the most widely grown vegetable crop in Delaware, and in 2018, the state led production nationally. A new disease on the crop is important to the agricultural economy of Delaware, and the yield loss potential of this pathogen needs assessment in the region. This species has only been reported in 2016 on lima bean, in New York (Gorny et al. 2016).

First Report of Colletotrichum nymphaeae Causing Anthracnose on Camellia oleifera in China

Camellia oleifera (tea-oil tree) is native to China and is cultivated in many parts due to its high-quality cooking oil (Liu et al. 2017; Ma et al. 2011). In February 2017, anthracnose lesions were found on leaves of C. oleifera planted in Guizhou province, China. The disease incidence was 20 to 30% in plantations with over 50 acres (20.2 ha) of tea-oil trees. The necrotic lesions on green leaves were surrounded by a ring of brown tissue and gradually turned gray. Three single-spore isolates of Colletotrichum-like fungus (GZGYYC6, GZGYYC7, and GZGYYC13) were isolated and cultured on potato dextrose agar (PDA) from surface-disinfested (75% ethanol and 0.5% v/v NaOCl) samples. Colonies of these cultures on PDA were gray, cottony in surface view, and pale gray to pale orange in reverse. The average mycelial growth rate was 9.3 mm per day (n = 5). Setae and sclerotia were absent. Conidia were smooth, hyaline, tapered to the base, rounded at the apex, and 6.5 to 19.6 × 2.0 to 4.8 μm (n = 200). These morphological characteristics correspond to those of Colletotrichum nymphaeae (Damm et al. 2012). Three single-spore isolates were used for molecular identification: internal transcribed spacer (ITS) and partial actin (ACT), β-tubulin 2 (TUB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genomic DNA regions were amplified by PCR and sequenced using the ITS5/ITS4 and ACT-512F/ACT-783R, T1/Bt2b, and GDF/GDR primer pairs, respectively (Weir et al. 2012). Gene sequences were deposited in GenBank (MF426212 to MF426214; MF426217 to MF426219; MF426232 to MF426234; MF426237 to MF426239). BLASTn analyses revealed that DNA sequences of our strains at the ITS, GAPDH, ACT, and TUB2 loci showed 99.8, 99.5, 100, and 100% sequence identity, respectively, to their corresponding loci in strains CBS129945 (GenBank accession no. MH865694), CBS126507 (JQ948533), Col-680 (MH717707), and BRIP62666 (KU221376) of C. nymphaeae. All four gene sequences were compared with representative sequences from type specimens or ex-type of Colletotrichum (Damm et al. 2012), and a phylogenetic tree was generated via the neighbor-joining method with 1,000 bootstrap replications using MEGA7 (Kumar et al. 2016). The tree placed the three isolates in the same group as C. nymphaeae (bootstrap 100%). Both morphological and molecular characteristics identified our isolates as C. nymphaeae. A pathogenicity assay was performed on three plants. Conidial suspension (10⁶ conidia/ml) of the representative isolate GZGYYC6 was dropped on three nonwounded and three wounded leaves, three wounded stems, and four wounded fruits per plant. Control leaves and stems were treated with distilled water. After inoculation, plants were kept under greenhouse conditions at 28 ± 2°C and 90% relative humidity on a 12-h fluorescent light/dark regimen. Anthracnose-like symptoms were observed 5 days postinoculation. The control plant tissues remained healthy. C. nymphaeae was reisolated on PDA from lesions, and the morphological features were consistent with GZGYYC6 isolate, confirming Koch’s postulates. To our knowledge, this is the first report of C. nymphaeae infection on C. oleifera in China. These findings will help in developing better preventive measures in accordance with the emergence of the new pathogen.

First Report of Black Dot Root Rot, Caused by Colletotrichum coccodes, on Tomato in Kentucky High Tunnels

As the most profitable vegetable crop grown in Kentucky (KY) high tunnels, tomato (Solanum lycopersicum L.) is often produced without crop rotation. In November 2016, ‘Mountain Fresh Plus’ tomato samples were received by the University of Kentucky Plant Disease Diagnostic Lab from Nelson County, KY. The high tunnel had an extended history of tomato production, and 100% of plants were affected. Common late-season symptoms of plant dieback and fruit decay were reported. Brown discoloration with dry, cracked cankers and microsclerotia were observed on primary and secondary sample roots, respectively. Symptomatic root sections (4 to 6 mm) were sterilized with 1% NaOCl and then placed on acidified potato dextrose agar (aPDA). Hyphal tips of the cultured fungus were aseptically transferred to fresh aPDA, and then mycelium was aseptically transferred to potato dextrose broth. DNA was extracted from 3-day-old broth culture (Li et al. 2008). Polymerase chain reaction (PCR) amplicons using ITS1/ITS4 (White et al. 1990) were generated, using protocols modified from Gardes and Bruns (1993) and Matsuda et al. (2005). Modifications included 35 total cycles, 30 s annealing step, and final extension for 10 min. Additional PCR products were amplified using partial actin (ACT; Prihastuti et al. 2009) and glyceraldehyde-3-phosphate dehydrogenase primers (GPDH; Berbee et al. 1999). ExoSap-IT (Affymetrix, Cleveland, OH) was used for PCR product cleanup, and then bidirectional Sanger sequence (Eurofin Genomics, Louisville, KY) was generated for each housekeeping gene. The internal transcribed spacer sequence was submitted to GenBank (MG544880) and matched 100% on identity and coverage with multiple Colletotrichum coccodes accessions, including KY364640. ACT and GPDH sequences were used in phylogenetic trees, which further supported identification as C. coccodes. The original isolate was grown on quarter-strength PDA at 23°C. Microsclerotia developed after 7 days within sparse, white mycelium. Conidia were dislodged from 4-week-old plates flooded with sterile deionized water using a sterile micropestle and then filtered through two layers of cheesecloth. Conidia were 18 to 22 μm long and 3 to 5 μm wide and were cylindrical, aseptate, and hyaline, with cylindrical conidiophores. Acervuli on tomato roots were circular with approximately 100-μm-long setae. Two-week-old ‘Sunstart’ tomatoes were used for Koch’s postulates. Eight plants were each drenched with 25 ml of 5 × 10⁵ conidia/ml (Vrisman et al. 2017). Eight plants drenched with 25 ml of sterile deionized water were noninoculated controls. After 11 weeks, root discoloration was observed, with black acervuli with setae visible on 50× magnified root surfaces; control plants were asymptomatic and lacked fungal growth. Symptomatic root cuttings were submerged in 0.6% NaOCl for 30 s and then plated on quarter-strength PDA. Mycelium was macroscopically visible after 1 week at ambient temperature. Microscopic examination indicated the fungus was morphologically indistinguishable from mycelium, conidiophores, and conidia of the original isolate, confirming Koch’s postulates. This experiment was repeated with the same results. Although C. coccodes and Pyrenochaeta lycopersici were recently reported to cause brown root rot complex in high tunnel tomato (Vrisman et al. 2017), P. lycopersici was not found here. This first report of black dot root rot in intensive high tunnel tomato production in KY reiterates the need for aggressive sanitation prior to disease development, because severe pathogen pressure in soil may necessitate relocation of the high tunnel.

Cladosporium omanense, a new endophytic species from Zygophyllum coccineum in Oman

The genus Cladosporium is cosmopolitan in distribution and can be isolated from various sources including plants as endophytes. A strain of Cladosporium was isolated from living leaves of Zygophyllum coccineum in a desert habitat in Oman. Phylogenetic analysis based on the combined internal transcribed spacer (ITS), partial translation elongation factor 1-alpha (TEF) and partial actin (ACT) sequence data shows that this is a novel species, which groups with Cladosporium sphaerospermum complex. The new species is introduced as Cladosporium omanense and is described, illustrated and compared with other known species in the genus. Cladosporium omanense differs from its phylogenetically related species C. endophyticum by its fast growing colonies, longer macronematous and micronematous conidiophores, thickened-walled conidia with conspicuously protuberant shrivelling surface ornamentations.

Hymenoscyphus pusillus, a new species on leaves ofFraxinus pennsylvanicain Poland

AbstractAmerican green ash (Fraxinus pennsylvanica) is among the most widely distributed ash species in North America. In the 18th century, it was introduced to Europe where it is used for ornamental purposes or for timber production. None of the currently knownHymenoscyphusspecies has been found onF. pennsylvanicain North America.Hymenoscyphus fraxineus, the cause of ongoing ash dieback epidemics, occurs onF. pennsylvanicain Europe but this species seems to show some degree of tolerance to the pathogen. A new species,Hymenoscyphus pusillus, detected inF. pennsylvanicaleaf litter in Poland is presented. The description is based on morphological and molecular studies.Hymenoscyphus pusillusproduces, in summer, whitish apothecia developing on black pseudosclerotia formed in petioles, rachises and leaf veins, in the litter of the previous year's leaf fall. The new species differs fromH. fraxineusin its smaller apothecia (up to 2.3 mm in diam.), absence of croziers at the ascus base, formation of superficial pseudosclerotia in the form of strips or isolated insular black areas on the thicker regions of rachises and petioles and rapidity of pseudosclerotia formation (6–8 weeks after leaf fall). The sizes of asci and ascospores ofH. pusillusare similar to those ofHymenoscyphus linearisand smaller than those of other species in theH. fraxineusgroup. Optimal temperature for growth ofH. pusillusin vitro is 20°C. The fungus does not produce an anamorph, distinguishing it fromH. fraxineus,H. linearisandHymenoscyphus occultus, which frequently formChalara‐like anamorphs. Analyses of sequence data for ITS rDNA and protein‐coding genes (partial actin, CAL and TEF1‐α) showed thatH. pusillusforms a well‐supported clade in the phylogenetic trees.Hymenoscyphus pusillusis closely related toH. occultus, detected onF. chinensissubs.rhynchophyllain Korea. After inclusion of the new species, theH. fraxineusgroup now consists of seven species. Some ecological and trophic aspects concerning the occurrence of the new species are discussed. The possibility of competition betweenH. pusillusandH. fraxineusis discussed.

First Report of Anthracnose on Michelia champaca Caused by Colletotrichum fioriniae in China.

Michelia champaca Linn., commonly named as yellow jade orchid tree, is best known for its strongly fragrant yellow or white flowers. It is primarily cultivated for its timber and also used in urban landscaping in China. Moreover, the flower buds are commonly used by many traditional healers in most of the herbal preparations for diabetes and kidney diseases (Jarald et al. 2008). In September 2017, necrotic spots were observed on young and mature leaves of M. champaca in Chongzhou County (30.63° N, 103.67° E), Sichuan Province, China. The symptoms initially appeared as small necrotic spots, circular to semicircular in shape, water soaked, later becoming grayish white in the center with a dark brown margin bordered by a tan halo. As the disease progressed, these spots coalesced and caused premature senescence and falling of the leaves, reducing their ornamental value. To identify the causal agent of leaf spots, small segments of symptomatic tissues were surface sterilized by washing them in 75% ethanol for 30 s and rinsing three times in sterile water. Tissues were placed on potato dextrose agar (PDA) at 25°C. A fungus was consistently isolated from symptomatic tissues on PDA. This fungus produced pale gray, dense aerial hyphae and black structures similar to sclerotia on PDA plates. The salmon-colored conidial mass was observed on well-developed colonies after 20 days. Acervuli were dark brown to black, 180 to 455 μm in diameter. Conidia were hyaline, aseptate, oblong, and 14.5 to 19.5 × 4.0 to 6.5 μm (n = 100). These morphological and cultural characteristics were consistent with the description of Colletotrichum spp. Isolate MC-2 was selected as a representative for molecular identification. The internal transcribed spacer (ITS) region of rDNA, partial actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), β-tubulin 2 (TUB2), and calmodulin (CAL) genes (Damm et al. 2012) were amplified and sequenced. Sequences were deposited in GenBank (MG657347 for ITS, MG657348 for ACT, MG657349 for GAPDH, MG657350 for CHS-1, MG657351 for TUB2, and MG657352 for CAL). BLAST analysis showed >99% identity with several reference sequences of C. fioriniae (Glomerella fioriniae) previously deposited in GenBank. Accordingly, the fungus isolated from anthracnose symptoms on M. champaca was identified as C. fioriniae. Three single-spore isolates were used for pathogenicity analyses. Twelve leaves of M. champaca were inoculated with a conidial suspension (5 × 10⁶ conidia/ml), with distilled water as a control. Small circular spots similar to those observed on naturally infected plants in the field were observed in all of the inoculated leaves within 8 days after inoculation. No symptoms were observed on the control. The inoculated fungus was reisolated from lesions on inoculated plant tissues and showed the same morphological and cultural properties as the original isolates. No C. fioriniae was isolated from control leaves, thus fulfilling Koch’s postulates. The results of the three repeated experiments were identical. To our knowledge, this is the first report of C. fioriniae causing anthracnose on M. champaca in China. This study provides useful information for the control of this disease in the future.

Novel Collophorina and Coniochaeta species from Euphorbia polycaulis, an endemic plant in Iran

During a study on the biodiversity of yeasts and yeast-like ascomycetes from wild plants in Iran, four strains of yeast-like filamentous fungi were isolated from a healthy plant of Euphorbia polycaulis in the Qom Province, Iran (IR. of). All four strains formed small hyaline one-celled conidia from integrated conidiogenous cells directly on hyphae and sometimes on discrete phialides, as well as by microcyclic conidiation. Two strains additionally produced conidia in conidiomata that open by rupture. The internal transcribed spacer (ITS) sequences suggested the placement of these strains in the genera Collophorina (Leotiomycetes) and Coniochaeta (Sordariomycetes), respectively. Blast search results on NCBI GenBank and phylogenetic analyses of ITS, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the translation elongation factor 1α (EF-1α) sequences, and the nuclear large subunit ribosomal gene (LSU), partial actin (ACT), and β-tubulin (TUB) sequences, respectively, revealed the isolates to belong to three new species, that are described here as Collophorina euphorbiae, Coniochaeta iranica, and C. euphorbiae. All three species are characterised by morphological, physiological, and molecular data.