Colletotrichum nanjingense sp. nov. and C. gloeosporioides s.s. Causing Leaf Tip Blight on Jasminum mesnyi in Nanjing, Jiangsu, China.

Jasminum mesnyi Hance is an important medicinal and ornamental plant. This species is native to South Central China and Vietnam and grows primarily in the subtropical biomes. In June 2022, 17 Colletotrichum strains were isolated from leaf tip blight on foliage of J. mesnyi in Nanjing, Jiangsu, China. Based on morphological characteristics and multilocus phylogenetic analyses of six genomic loci (ITS, CAL, ACT, TUB2, CHS-1, and GAPDH), a new species, namely, C. nanjingense, and a known species, namely, C. gloeosporioides s.s., were described and reported. Pathogenicity tests revealed that both species were pathogens causing leaf tip blight on J. mesnyi. The results provided necessary information for disease control and enhanced our understanding of the diversity of Colletotrichum species in China.

First report of Neopestalotiopsis clavispora causing leaf spot on Phoebe bournei in China.

Phoebe bournei (Hemsl.) Yang is a typical evergreen broadleaf species widely distributed in subtropical China for its ornamental and economic value (Zhang et al. 2021). The wood of P. bournei is considered a good material for architectural decoration and furniture (Li et al. 2018). In June 2020, leaf spot symptoms were observed in Dexing (28°41'22.056″N, 115°51'52.524″E), Jiangxi province, China. Initial disease symptoms were small brown spots on the leaves. Then, the spots enlarged and coalesced into regular or irregular dark brown necrotic lesions with dark margins. Disease incidence in the field in Dexing was estimated 25%. Leaf pieces (5 × 5 mm) from the lesion borders were surface-sterilized in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C under a 14/10 h light/dark cycle for 4 days. Pure cultures were obtained by monosporic isolation, and the representative isolates, JX-N2, JX-N7, and JX-N11 were used for morphological studies and phylogenetic analyses. The colonies of three isolates grown on PDA were white, cottony, and flocculent, contained undulate edges with dense aerial mycelium on the surface. Conidia were 5-celled, clavate to fusiform, smooth, 18.7-24.6 × 5.9-8.8 µm (n = 100). The 3 median cells were dark brown to olivaceous, central cell was darker than other 2 cells, and the basal and apical cells were hyaline. All conidia developed one basal appendage (3.4-8.3 µm long; n = 100), and 2-3 apical appendages (17-30 µm long; n = 100), filiform. Morphological features were similar to Neopestalotiopsis sp. (Maharachchikumbura et al. 2014). The internal transcribed spacer (ITS) regions, ß-tubulin 2 (TUB2) and translation elongation factor 1-alpha (TEF1-α) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, T1/Bt-2b, EF1-728F/EF-2 (Maharachchikumbura et al. 2014), respectively. All sequences were deposited into GenBank (ITS, OQ355048 - OQ355050; TUB2, OQ357665 - OQ357667; TEF1-α, OQ362987 - OQ362989). A maximum likelihood and Bayesian posterior probability-based phylogenetic analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (ITS, TUB2, TEF1-α) placed JX-N2, JX-N7, and JX-N11 in the clade of N. clavispora. Based on the multi-locus phylogeny and morphology, the representative isolates were identified as N. clavispora. The pathogenicity of three isolates were tested on six 9-year-old P. bournei plants, which were grown in the field. Three leaves per plant were wounded with a sterile needle (Φ=0.5 mm) and inoculated with 20 µL conidial suspension per leaf (106 conidia/mL). Another six control plants were inoculated with sterile water. Each leaf was covered with plastic bags to keep a humidity environment for 2 days. All the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 9 days. N. clavispora was reisolated from the lesions, whereas no fungus was isolated from control leaves. N. clavispora can cause leaf diseases in a variety of hosts, including Machilus thunbergii (Wang et al. 2019), Fragaria × ananassa (Shi et al. 2022), Taxus media (Li et al. 2022). However, this is the first report of N. clavispora infecting P. bournei in China. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

First report of Diaporthe acuta causing leaf blight of Acer palmatum in China.

Acer palmatum Thunb. is an important ornamental deciduous tree with colorful foliage, and widely cultivated in Japan, Korea and China (Carlos et al. 2016). In October 2021, a foliar disease of ~95% incidence was observed on A. palmatum in three community parks, Shaoxing, Xuzhou, and Wuhan cities, China. The symptoms appeared as brown necrotic lesions at the tips, margin, and surface of leaves. Thirty leaves with symptoms from three trees were collected from the three parks. Small pieces (3 to 5 mm2) cut from the lesion margins were placed on potato dextrose agar (PDA) after surface-sterilized and incubated at 25°C in the dark, following the protocol described previously (Wan et al. 2022). The same fungus was isolated from 31% of 150 tissue pieces. Pure cultures were obtained from the tip of hyphae. Three representative isolates (WH52, SX13, and XZ96) were obtained and deposited at Nanjing Forestry University. The colony on PDA was white with aerial mycelia, cottony, and the reverse was white. Gray pycnidia developed on the sterile alfalfa stems at 25°C with a 14/10 h light/dark cycle in 30 days. Conidiophores were hyaline, cylindrical, septate, branched, smooth, 14.3-37.2 × 1.5-3.7 µm (n = 30). Conidiogenous cells were cylindrical, 5.6-21.6 × 1.3-2.1 µm (n = 30). Alpha conidia were aseptate, fusiform to oval, 6.5 ± 0.6 × 2.2 ± 0.2 µm (n = 50), bi- or multi-guttulate. Beta conidia were aseptate, hyaline, and curved, 31.0 ± 3.5 × 1.0 ± 0.1 µm (n = 30). Gamma conidia were aseptate, infrequent, botuliform, 12.4 ± 1.2 × 1.4 ± 0.1 µm (n = 10). Morphological characteristics of the three isolates matched those of Diaporthe spp. (Gomes et al. 2013). DNA of the three isolates was extracted and the internal transcribed spacer region (ITS), histone H3 (HIS), partial translation elongation factor 1-alpha (TEF1-α), beta-tubulin (TUB), and calmodulin (CAL) genes were amplified with primers ITS1/ITS4 (White et al. 1990), CYLH3F/H3-1b (Glass and Donaldson, 1995; Crous et al. 2004), EF1-728F/EF1-986R (Carbone et al. 1999), Bt2a/Bt2b (Glass and Donaldson 1995), and CAL-228F/CAL-737R (Carbone et al. 1999), respectively. The genomic DNA sequences were deposited in GenBank with Accession Nos. OP522005, OP522447, OP522448, and OP566419 to OP566430 (Supplementary Table 1). BLAST search of the sequences from the three isolates showed high similarities with sequences of Diaporthe acuta Y.S. Guo & G.P. Wang (ex-type PSCG 047). BLAST results were listed in Supplementary Table 1. Maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and MrBayes v. 3.2.6 with the concatenated sequences placed WH52, SX13, and XZ96 in the clade of D. acuta. Based on the phylogeny and morphology, the three isolates were identified as D. acuta. The pathogenicity was tested on potted 3-yr-old seedlings of A. palmatum. Healthy leaves wounded with a sterile needle (1 mm in diameter) were inoculated with 5-mm plugs from the edge of 3-day-old culture of the three isolates. The PDA plugs were used for controls. Three plants were used for each treatment, and three leaves of each plant were inoculated. Each plant was covered with a plastic bag, and sterilized water was sprayed into the bags to maintain humidity in a greenhouse at the day/night temperatures at 25 ± 2°C. The plastic bags were removed on the fifth day. Five days after inoculation, the inoculated leaves appeared lesions similar to those in the field. The controls remained healthy. Diaporthe acuta was reisolated from the lesions on the inoculated leaves and was confirmed based on morphological characteristics and ITS sequence analyses. No fungus was isolated from the controls. Diaporthe acuta was previously reported to cause pear shoot canker in China (Guo, et al. 2020), and D. foliicola, D. monospora and D. nanjingensis caused leaf blight of A. palmatum (Wan et al. 2022). This is the first report of D. acuta causing leaf blight of A. palmatum. This finding will provide an effective basis for developing control strategies for the disease.

Fusarium pernambucanum Causing Leaf Yellow Spot on Melon (Cucumis melo L.), a New Disease in China.

Melon (Cucumis melo L.) is a member of the Cucurbitaceae family, and is an important economic and horticultural crop. In March 2022, melon plants in greenhouses exhibited severe leaf yellow spot symptoms in Changjiang County (109°13'N, 19°28'E), Hainan Province. The incidence of the disease was about 30-50%. Lesions initially appeared as yellow dots on leaves and expanded irregularly. Gradually, brown spots appeared, and finally the whole leaves turned yellow and resulted in blighting and death of foliage (Figure 1.). A total of four symptomatic plants were sampled from about 0.2 ha of an area. Symptomatic leaves were excised, surface disinfected with 2% (w/v) NaOCl, rinsed three times with sterile distilled water, and placed on potato dextrose agar (PDA) followed by incubation at 25°C in the dark for 5 days. The pure cultures were obtained by the hyphal-tip method. A total of eight fungal isolates with similar colony characteristics were recovered from the four symptomatic plants. Three DNA fragments (ITS, TEF1, and RPB2) of the eight isolates showed 100% sequence identity based on the molecular identification methods described below. Therefore, one of the isolates, M2JP-3, was chosen for identification and test of the pathogenicity. The colony of M2JP-3 on PDA at 25°C for 5 days was white with yellow-brown pigmentation in the center (Figure 2A-B). From 10-day-old cultures grown on CLA (Fisher et al. 1982), macroconidia (n = 50) were falcate, slender, curved dorsiventrally, tapering towards both ends, 3 to 7 septate, and measured 24.5 to 52.1 x 3.7 to 4.7 µm. The microconidia (n = 50) were straight or slightly curved, septate 0 to 2, and measured 9.9 to 16.3 x 2.5 to 3.7 µm (Figure 2C-E). For molecular identification, genomic DNA was extracted using the method previously described (Khan et al. 2021),the internal transcribed spacer (ITS), translation elongation factor 1α (TEF1) and DNA-dependent RNA polymerase subunit II (RPB2) were amplified, respectively, using primers ITS1/ITS4 (White et al. 1990), EF1/ EF2 (O'Donnell et al. 1998), and 5F2/7cR (Reeb et al. 2004). The 529 bp (ITS), 723 bp (TEF1), and 965bp (RPB2) sequences were deposited in GenBank with acce. nos. OP303211, OP312675 and OP312674, respectively. A phylogenetic tree was constructed using the concatenated three gene sequences of M2JP-3 and that of the Fusarium incarnatum-equiseti species complex (FIESC) (Xia et al. 2019) based on Maximum Likelihood (Figure 3). M2JP-3 was grouped together with the F. pernambucanum strain NRRL 32864 (accession no. GQ505702 for ITS, GQ505613 for TEF1and GQ505791 for RPB2), and shared 100% concatenated sequence identity. For pathogenicity tests of M2JP-3, seeds of melon cultivar Jinmeiren were surface disinfected and sowed in soil in three replicated pots in a greenhouse at 26 °C under natural light. Healthy leaves of the melon plants were wounded with needles and inoculated with mycelial plugs of M2JP-3 or PDA plugs as control. . Symptoms similar to the original greenhouse symptoms were observed at 7 days after inoculation (Figure 4). The control leaves were asymptomatic. The same fungus was reisolated from the inoculated leaves, as identified based on morphology and molecular evidence, which confirmed the Kochs' postulates. To our knowledge, this is the first time Fusarium pernambucanum has been recorded causing leaf yellow spot disease on melon. Further, findings of the present study will help to develop effective disease management strategies against Fusarium pernambucanum Leaf Yellow Spot on melon in China.

First report of leaf spot and stem blight on blueberry (Vaccinium corymbosum cv. Bluerain) caused by Calonectria pseudoreteaudii in China.

Blueberry has high nutritional value and is one of the five healthy fruits. In 2018, leaf spots and stem blights were observed on Vaccinium corymbosum cv. Bluerain in Guangzhou, Guangdong Province, China. Up to 80% of the plants were affected. Initial symptoms of affected leaves were red-brown, irregular, small spots, which gradually coalesced and formed larger irregular necrotic patches. The affected stems showed red-brown and irregular large lesions. Diseased tissues were surface sterilized with 75% alcohol for 15 s, followed by 2.5% NaClO for 30 s, and rinsing three times in sterile distilled water, placed on potato dextrose agar (PDA) and incubated at 25 C. Representative strains, ZHKUCC 21-0021 from diseased leaves and ZHKUCC 21-0073 from diseased stems, were selected for further studies. Colonies grew slowly at 25 C on malt extract agar (MEA) (average 5.68 mm/d), producing white aerial mycelium and red-brown color on the underside after 7 days. Macroconidiophores were hyaline, smooth, consisting of a stipe bearing fertile branches, and a stipe extension terminating in a vesicle. Each terminal branch produced 2-4 phialides, 8-13 × 3-6 µm, reniform or doliiform; Stipe extensions were septate, terminating in a narrowly clavate vesicle, 2-6 µm. Macroconidia were hyaline, straight cylindrical, round at both ends, 83-100 × 7-11 µm (average = 94 × 8 µm; n = 50), with 5 septa. These morphological characteristics were similar to the description of Calonectria pseudoreteaudii (Lombard et al., 2010). The partial calmodulin (cmdA), beta-tubulin (ß-tubulin), and translation elongation factor 1-alpha (tef1-α) genes of the two isolates were respectively amplified using primers CAL-228F/CAL-737R (Carbone et al., 1999), EF1-728F/EF2 and T1/CYLTUB1R (Lombard et al., 2015), and sequences were deposited in GenBank (cmdA: MZ516854 and MZ516855; ß-tubulin: MZ516858 and MZ516859; tef1-α: MZ516856 and MZ516857). BLAST analysis of three gene sequences showed 100% similarity to those of C. pseudoreteaudii. In the maximum likelihood (ML) tree of the concatenated sequences of the three genes, the two isolates from this study were clustered with C. pseudoreteaudii with 100% bootstrap support. Five-mm-diameter hyphal plugs of two representative isolates grown on PDA for five days were used in the pathogenicity test. Leaves were inoculated with ZHKUCC 21-0021, and stems were inoculated with ZHKUCC 21-0073 with five replicates. As controls, sterile PDA plugs were used. All inoculated plants were maintained at 25 C . After 7 days, inoculated leaves and stems developed symptoms similar to field samples, whereas the control plants remained asymptomatic. The pathogen was reisolated from inoculated plants and confirmed to be C. pseudoreteaudii by morphological characteristics. Five Calonectria species (C. canadensis, C. colhounii, C. ilicicola, C. kyotensis and C. pyrochroa), have been reported associated with blueberry (Farr and Rossman, 2022; Fei et al, 2017). Calonectria canadensis and C. ilicicola have been reported to cause stem blight and stem rot in Vaccinium spp. in China (Fei et al, 2017 and 2018). Calonectria colhounii has been reported to cause stem blight in V. angustifolium and V. corymbosum in the United States (Sadowsky et al, 2011). However, this is the first report of C. pseudoreteaudii causing leaf spot and stem blight on Vaccinium spp. worldwide. These results will provide a foundation for future research on prevention and control of this disease.

Diaporthe sapindicola sp. nov. Causes Leaf Spots of Sapindus mukorossi in China.

Sapindus mukorossi Gaertn. (Sapindaceae), or soapberry, is an important biodiesel tree in southern China. In recent years, leaf spot disease on soapberry has been observed frequently in a soapberry germplasm repository in Jianning County, Sanming City, Fujian province, China. The symptoms initially appeared as irregular, small, yellow spots, and the centers of the lesions became dark brown with time. Three fungal isolates from lesions were collected. Koch's postulates were performed, and their pathogenicity was confirmed. Morphologically, α-conidia from diseased tissues were single-celled, hyaline, smooth, clavate or ellipsoidal, and biguttulate, measuring 6.2 to 7.2 × 2.3 to 2.7 µm. In addition, the three isolates in this study developed three types (α, ß, and γ) of conidia on potato dextrose agar, and their morphological characteristics matched those of Diaporthe. A phylogenetic analysis based on internal transcribed spacer, TEF, TUB, HIS, and CAL sequence data determined that the three isolates are a new species of Diaporthe. Based on both morphological and phylogenetic analyses, the causal fungus, Diaporthe sapindicola sp. nov., was described and illustrated.

First Report of Rose Apple Leaf Spot Caused by Colletotrichum siamense in Thailand.

The rose apple (Syzygium samarangense (Blume) Merr. & L.M.Perry) plant has been commonly cultivated in Thailand. In May of 2022, leaf spot disease of rose apple was discovered in Chiang Mai Province, Thailand, with approximately 30% disease incidence. The typical symptoms initially showed brown spots (0.1 to 0.5 mm in diameter) with a yellow halo surrounding. These spots then expanded with black edges and the infected leaves appear blighted and desiccated. In humid conditions, pale yellow conidiomata formed on the lesions. Small pieces (5 × 5 mm2) of the margins between lesions and the healthy tissue were surface disinfected with 1% NaClO for 1 min, 70% ethanol for 30 s, and washed three times with sterile distilled water. Tissues were placed on potato dextrose agar (PDA) and incubated at 25 ºC for three days. Three fungal isolates (SDBR-CMU419, SDBR-CMU420, and SDBR-CMU421) were obtained that exhibited similar morphology. Fungal colonies appeared white to gray with cottony mycelia after incubation on PDA at 25 ºC for one week. All fungal isolates produced asexual morph on PDA. Setae were 5590 × 2.53.5 µm, brown with 13-septa, cylindrical base, and tip rounded. Conidiophores were hyaline to pale brown, septate, and branched. Conidiogenous cells were hyaline to pale brown, cylindrical to ampulliform, 2050 µm long (n = 50). Conidia were one-celled, hyaline, smooth-walled, aseptate, straight, cylindrical, end round, guttulate, 1017 × 35 µm (n = 50). Appressoria were mostly formed from mycelia, oval to irregular, brown to dark brown, smooth-walled, 610 × 57 µm (n = 50). Morphologically, all fungal isolates resembled to Colletotrichum (Weir et al. 2012; Jayawardena et al. 2021). The internal transcribed spacer (ITS) region of the ribosomal DNA, actin (act), ß-tubulin (tub2), calmodulin (CAL), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified using primer pairs ITS5/ITS4 (White et al. 1990), ACT-512F/ACT-783R (Carbone and Kohn 1999), T1/T22 (O'Donnell and Cigelnik 1997), CL1C/CL2C (Weir et al. 2012), and GDF1/GDR1 (Templeton et al. 1992), respectively. The ITS (ON740892 to ON740894), act (ON759242 to ON759244), tub2 (ON759245 to ON759247), CAL (ON759248 to ON759250), and GAPDH (ON759251 to ON759253) sequences were deposited in GenBank. Multi-gene (combined data set of ITS, GAPDH, CAL, act, and tub2) maximum phylogenetic analyses indicated that all fungal isolates clustered with C. siamense ICMP 18578 (type strain) with strong statistical (99% ML) support. For pathogenicity test, asymptomatic leaves, stems and fruits detached from healthy plants were surface disinfected using 0.1% NaClO for 3 min, washed three times with sterile distilled water, and air-dried. A uniform wound (3 pores, 1 mm in width) was made at the equator of each leaf, stem and fruit using aseptic needles. Mycelial plugs (5 mm in diameter) and conidia suspensions (1 × 106 conidia/ml) of each fungal isolate grown on PDA at 25 ºC for one week were used to inoculate both wounded and unwounded samples by the detached method (Huda­Shakirah et al. 2022; Suwannarach et al. 2022). Plugs of PDA and sterile distilled water were used as controls. Ten replications were performed for each treatment and the experiment was repeated twice. All inoculated samples were incubated in a moist chamber at 25 ºC with 90% relative humidity. The disease severity index was used to evaluate the specimens (Acar et al. 2008; Ngegba et al. 2017). After one week, both wounded and unwounded leaves that inoculated with mycelial plugs and conidia suspensions showed brown leaf spots and a weak infection. Mycelial plugs inoculated on both wounded and unwounded fruits revealed a moderate infection, but inoculation of conidia suspensions showed a weak infection. No symptoms of disease were observed on the inoculated stems. Control leaves, stems and fruits remained asymptomatic. The pathogen C. siamense was re-isolated from spot and rot lesions on PDA in order to fulfill Koch's postulates. Phoulivong et al. (2012) reported that C. siamense is a causal agent of fruit rot in rose apples cultivated in Lao and Thailand. To our knowledge, this is the first report of C. siamense causing leaf spots on rose apple plants in Thailand. Importantly, these findings will provide crucial information for epidemiologic studies and in the development of appropriate management strategies for this newly emerging disease.

First report of Erysiphe corylacearum Causing Powdery Mildew on Hazelnut in Hungary.

The interest in hazelnut (Corylus avellana L.) cultivation has recently increased in Hungary, it is currently grown on 490 hectares. In August 2021 early powdery mildew symptoms were observed in a hazelnut plantation, and in a variety collection of the Hungarian University of Agricultural and Life Sciences in Érd. White patches of mycelium and conidia were observed on both side of the leaves. In early October abundant chasmothecia were formed. The disease incidence was 100% on varieties 'Segorbe', and 'Corabel', 70% on 'Ennis', and 30% on the leaves of 'Istrska dolgoplodna leska' (15 plants per cultivar). Powdery mildew is usually caused by Phyllactinia guttata, which was present abundantly on the abaxial and sparsely on the adaxial surface of the observed leaves. However, another fungus co-occurred on the adaxial surface of the leaves, and rarely occurred on the abaxial surface of the leaves. Its morphology differed substantially from P. guttata on having smaller chasmothecia, and branched appendages. The new powdery mildew agent was morphologically described. Mycelium was hyaline, branched, septate, thin-walled and smooth, 2.5-3.1 µm wide. Conidiophores measured 22 to 61 × 5.1 to 8.5 (average: 44.1 × 6.5) µm (n = 30), the foot cells were erect, cylindrical, and flexuous. Conidia occurred rarely and were produced single on conidiophores, 19 to 34 × 15 to 24 µm. Chasmothecia were spherical, 74 to 103 (average: 85) µm in diameter (n = 100), single or in groups on both sides of each leaf. Appendages 7 to 15 per chasmothecium, aseptate, straight, sometimes flexuous with a length of 74 to 118 (average: 103) µm (n = 50), and had 3 to 5 times dichotomous branched apices with curved tips. Each chasmothecium contained 3 to 5 asci. Ovoid to subglobose asci measured 43 to 65 × 32 to 54 µm (average: 56 × 43) µm (n = 30). Asci contained 4 to 8 ascospores which were hyaline, ellipsoid, measured 17 to 23 × 11 to 20 (mean: 21 × 15) µm (n = 40) in diameter. Morphological identification was confirmed by molecular analysis of two samples, one from the plantation, and one from the variety collection. After DNA extraction partial rDNA internal transcribed spacer region (ITS) of the isolates was amplified using primers ITS1_F and ITS4_R, as previously indicated (Meparishvili et al. 2019). Obtained sequences were deposited to the GenBank (accession no. OL744964 and OL744961). BLAST analysis indicated that the two samples were showing 100% and 97,81% identity to ITS rDNA sequences of Erysiphe corylacearum from Switzerland (MN822722), and showed low similarity of 83% and 85% each to P. guttata (AB080558). Pathogenicity tests were accomplished on ten healthy two-year-old plants of C. avellana cv. 'Merveille de Bollwiller' with the two isolates under controlled environment on 25°C, 80% humidity and 16/8 photoperiod. Plants were artificially inoculated by conidial suspension droplets (104/ml). Symptoms appeared after 7-8 days after inoculation and the developing fungus was morphologically identical to the original isolates. Control plants were treated with distilled water, no symptoms were found on them. E. corylacearum was first observed on C. avellana in Turkey in 2013 (Sezer et al. 2017) and was considered as a highly destructive pathogen. It is also known in neighbouring countries, Ukraine (Heluta et al, 2019), Austria (Voglmayr et al. 2020) and Romania (Rosati et al. 2021). To our knowledge, this is the first report of Erysiphe corylacearum in Hungary.

First report of anthracnose on Acer fabric caused by Colletotrichum siamense in China.

Acer fabri Hance, an evergreen tree, is widely cultivated in China for its ornamental value (Lin. 2020). In July 2020, a leaf spot disease, with an incidence of Approximately 48% (12 out of 25), was observed on A. fabri plants (almost 9-year-old) at the campus of Jiangxi Agricultural University (28°45'56″N, 115°50'21″E). On average, 30% of the leaves per individual tree were affected. Small spots initially formed along the edge or tip of the leaves and gradually expanded into dark brown spots, and eventually the diseased leaves withered. Leaf pieces (5 × 5 mm) from the lesion borders were surfaced sterilized in 70% ethanol for 30 s, followed by 2% NaOCl for 1 min, and then rinsed three times with sterile water (Wan et al. 2020). Tissues were placed on potato dextrose agar (PDA) and incubated at 25°C. Pure cultures were obtained by monosporic isolation, and the representative isolates, LFY-1, LFY-5, and LFY-8 were used for morphological studies and phylogenetic analyses. Colonies on PDA of the three isolates were white to gray with cottony mycelia and grayish-white on the undersides of the culture. Conidia were single-celled, straight, hyaline, cylindrical, clavate, and measured 12.8-17.4 ×4.3-5.7 µm (14.3 ± 1.1 × 4.6 ± 0.4 µm, n = 100). Appressoria were brown to dark brown, ovoid to clavate, slightly irregular to irregular, and ranged from 5.6-9.3 × 4.7-6.6 µm (7.4 ± 0.3 × 5.5 ± 0.4 µm, n=100). Morphological features were similar to Colletotrichum gloeosporioides species complex (Weir et al. 2012). The internal transcribed spacer (ITS) regions, actin (ACT), calmodulin (CAL), beta-tubulin 2 (TUB2), chitin synthase (CHS-1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were amplified from genomic DNA for the three isolates using primers ITS1/ITS4, ACT-512F/ACT-783R, CL1/CL2, T1/Bt2b, CHS-79F/CHS-354R and GDF/GDR (Weir et al. 2012), respectively. All sequences were deposited into GenBank (ITS, OL818322- OL818324; ACT, OL830175 - OL830177; GAPDH, OL830166 - OL830168; TUB2, OL830163 - OL830165; CHS-1, OL830169 - OL830171; CAL, OL830172 - OL830174). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences placed LFY-1, LFY-5, and LFY-8 in the clade of C. siamense. Based on the multi-locus phylogeny and morphology, three isolates were identified as C. siamense. The pathogenicity of three isolates was tested on six A. fabri plants, which were grown in the field. Healthy leaves were wounded with a sterile needle and inoculated with 10 µL of spore suspension (106 conidia/mL). The spore suspension of each isolate was inoculated onto five leaves. Another three plants inoculated with ddH2O served as the control (Si et al. 2019). All the inoculated leaves were covered with plastic bags to keep a high-humidity for 2 days. All the inoculated leaves showed similar symptoms to those observed in the field, whereas control leaves were asymptomatic for 8 days. C. siamense was reisolated from the lesions, whereas no fungus was isolated from control leaves. The pathogen was previously reported to cause anthracnose on Kadsura coccinea (Jiang et al. 2022), Carica papaya (Zhang et al. 2021), Michelia alba (Qin et al. 2021). This study is the first to report C. siamense causing anthracnose on A. fabric. This work provided crucial information for epidemiologic studies and appropriate control strategies for this newly emerging disease.

First Report of Top Blight of Cunninghamia lanceolata Caused by Diaporthe unshiuensis and Diaporthe hongkongensis in China.

Cunninghamia lanceolata (Lamb.) Hook. is an important conifer species widely planted in southern China. A top blight, with an incidence of 20% (40/200 seedlings), occurred on 1-year-old seedlings of C. lanceolata in a nursery, Luzhai, Guangxi, China in August 2021. The disease mainly occurred on shoot tips. The infected needles and shoots appeared brown to brownish red. White conidial tendrils oozed from pycnidia under wet-weather conditions. Lesion margins from fresh samples were cut into small pieces (n=100), which were sterilized according to Mao et al., and placed on potato dextrose agar (PDA) at 25°C. Three isolates (GXJ2, GXJ4, and GXJ6) were obtained and deposited in The China Forestry Culture Collection Center (CFCC 55717, CFCC 55716, and CFCC 55722). The colony of GXJ2 on PDA was white, with sparse aerial mycelia, and became grey with time. The α conidia were fusiform, hyaline, and aseptate, 6.7±0.6 µm × 2.6±0.2 µm (n=30). The ß conidia were filiform, hyaline, and curved, 30.4±2.1 µm × 1.4±0.1 µm (n=30). Colonies of GXJ4 and GXJ6 were white, with moderate aerial mycelia, which collapsed at the center, and the collapsed parts were iron-gray. The α conidia were 7.8±0.8 µm × 2.5±0.2 µm (n=30). The ß conidia were absent. Morphological characters of 3 isolates matched those of Diaporthe spp.. The partial sequences of ITS, EF1-α, CAL, ß-tub, and HIS genes were amplified with primers ITS1/ITS4, EF1-728F/EF1-986R and CAL228F/CAL737R, ßt2a/ßt2b and CYLH3F/H3-1b according to Gomes et al. 2013, respectively. The sequences for the five genes of each of 3 isolates were deposited in GenBank (Accession Nos. see Supplementary Table 1). BLAST results showed that the ITS, EF1-α, ß-tub, HIS, and CAL sequences of GXJ2 were highly similar (>99%) with sequences of Diaporthe unshiuensis, while sequences of GXJ4 and GXJ6 were highly similar (>99%) to those of D. hongkongensis (Supplementary Table 1). Phylogenetic analyses using concatenated sequences placed GXJ2 in the clade of D. unshiuensis, while GXJ4 and GXJ6 in the clade of D. hongkongensis. Based on the phylogeny and morphology, GXJ2 was identified as D. unshiuensis, GXJ4 and GXJ6 as D. hongkongensis. Pathogenicity tests were performed on nine 1-year-old seedlings of C. lanceolata, and 10 needles at shoot tip per seedling were slightly wounded and inoculated with 5-mm mycelial plugs from one of 3 isolates. Three control seedlings were treated with PDA plugs. Each plant was covered with a plastic bag after inoculation and kept in an air-conditioned nursery at 25°C/16°C (day/night). The symptoms appeared 5-8 days after inoculation and were similar to those observed in the nursery. D. unshiuensis and D. hongkongensis were re-isolated from the inoculated seedlings and were confirmed based on morphology and ITS sequences. The controls were symptomless, and no fungus was isolated from them. D. unshiuensis was first reported as an endophyte on the fruit of Citrus unshiu, and caused peach constriction canker, shoot blight of kiwifruit. D. hongkongensis was first described from fruit of Dichroa febrifuga and caused shoot canker of pear, shoot blight and leaf spot of kiwifruit, and fruit rot of peach. This is the first report of D. unshiuensis and D. hongkongensis causing the top blight of C. lanceolata. This study provides a basis for controlling this newly emerging disease in the nursery.

First Report of Diaporthe eres and D. unshiuensis causing Leaf Spots on Sapindus mukorossi in China.

Sapindus mukorossi Gaertn., commonly known as soapberry, is widely cultivated as a landscaping tree in Southern China. In June 2019, a foliar disease with an incidence of ∼60% occurred on trees was observed in the soapberry germplasm repository, Jianning, Sanming, Fujian, China. The symptoms initially appeared as irregular small yellow spots, while the center of the lesions became dark brown with time. Fragments (size 3 to 4 mm2) taken from lesion margins were sterilized and cultured based on Wang et al. Two isolates (FJ1 and FJ21) were obtained with the following morphological characteristics on PDA, (1) FJ1: Conidiogenous cells were 9.7 to 25.0 × 1.5 to 2.2 µm (n=20). Alpha conidia were 6.1 to 8.3 × 2.2 to 3.0 µm (n=30), aseptate, hyaline, smooth, ellipsoidal. Beta conidia were 28.3 to 38.2 × 1.3 to 1.7 µm (n=30), hyaline, smooth, curved to hooked. Conidial drops were milky colored; (2) FJ21: Pycnidia were dark brown, 280 to 843 µm (n=30) in diam., globose, or irregular on alfalfa stems. Conidiophores were hyaline, cylindrical, smooth, and slightly tapered to the apex, 17.4 to 35.4 × 1.5 to 2.6 µm (n=20). Conidiogenous cells were 14.7 to 29.7 × 1.4 to 2.6 µm (n=20). Alpha conidia were 5.6 to 7.1 × 2.4 to 3.4 µm (n= 30), hyaline, smooth, ellipsoidal, or clavate, aseptate, biguttulate. Beta conidia not observed. Conidial drops were yellow. The morphological characteristics of FJ1 and FJ21 were similar to those of Diaporthe spp.. DNA of two isolates was extracted, and the internal transcribed spacer region (ITS) and partial sequences of translation elongation factor 1-alpha (TEF1-α), calmodulin (CAL), ß-tubulin (TUB), and histone H3 (HIS) genes were amplified with primers ITS1/ITS4, EF1-728F/EF1-986R, CAL228F/CAL737R, ßt2a/ßt2b, and CYLH3F/H3-1b, respectively. The sequences were deposited in GenBank (accession nos. MW585608 and MW768905 to MW768908 for FJ1; MT755625 and MT776728 to MT776731 for FJ21). The BLASTn results showed that the ITS, TEF1-α, TUB, HIS, and CAL sequences of FJ1 were 100, 99, 98, 98, and 99% identical to those of D. eres (NR144923, KJ210550, KJ420799, KJ420850, and KJ434999, respectively). For FJ21, BLASTing with the same loci showed 100, 100, 100, 99, and 100% similarity with those of D. unshiuensis (MH121530, MH121572, MH121607 MH121488, and MH121448, respectively). Phylogenetic analyses with the concatenated sequences placed FJ1 and FJ21 in the clades of D. eres and D. unshiuensis, respectively. Pathogenicity tests were performed by wounding leaves of 2-year-old soapberry seedlings with a sterile needle. The leaves were inoculated with D. eres and D. unshiuensis isolates, respectively, with 10 µl of conidial suspensions (106 conidia/ml). Three plants were used for each treatment, and the leaves of each plant were inoculated. The control was treated with 10 µl of sterile water. The plants were kept in a greenhouse (RH > 80%, 25 ± 2°C). In 5 days, all inoculated leaves showed lesions similar to the field symptoms. Controls were asymptomatic. Diaporthe eres and D. unshiuensis were reisolated from the diseased leaves. No fungus was isolated from the control. Previously, D. biconispora and D. sapindicola were reported as the causal agents of soapberry, but this is the first report of D. eres and D. unshiuensis causing leaf spots on S. mukorossi in China. These data will help develop effective strategies for managing this disease.

First Report of Diaporthe eres Causing Leaf Spot of Viburnum odoratissimum var. awabuki in China.

Viburnum odoratissimum var. awabuki (K. Koch) Zabel ex Rumpl. is an evergreen tree, used as a landscape plant in China. In June 2019, a foliar disease of ~60% incidence was observed on V. odoratissimum var. awabuki at the campus of Nanjing Forestry University, Jiangsu, China. The symptoms were initially irregular small red-brown spots, later enlarged and became brown to black. Small pieces of tissue (3 to 4 mm2) cut from lesion margins were surfaced sterilized in 75% ethanol for 30 s and 1.5% NaClO for 60 s, then rinsed in sterile water and placed on potato dextrose agar (PDA) at 25℃. Pure cultures were obtained from the tip of hyphae. Using the standard phytopathological procedure, two representative isolates (SH161 and SH181) were obtained and deposited at Nanjing Forestry University. The colony on PDA was white with aerial mycelium, radiate, and the reverse was white. Black pycnidia developed on the sterilized alfalfa stems at 25°C with a 14/10 h light/dark cycle for 20 days. Conidiophores were hyaline, branched, straight to sinuous, 9.4 to 26.0 × 1.0 to 2.5 µm (n=30). Conidiogenous cells were 2.1 to 15.1 × 0.9 to 2.5 µm (n=30). Alpha conidia were 7.4 ± 0.6 × 2.0 ± 0.2 µm (n=50), hyaline, ellipsoidal to lanceolate. Beta conidia were 29.5 ± 1.8 × 1.1 ± 0.1 µm (n=30), aseptate, hyaline, smooth, curved to hooked. Morphological features of two isolates matched those of Diaporthe spp.. DNA of two isolates was extracted and the internal transcribed spacer region (ITS), partial translation elongation factor 1-alpha (TEF1-α), calmodulin (CAL), beta-tubulin (TUB), and histone H3 (HIS) genes were amplified with primers ITS1/ITS4, EF1-728F/EF1-986R, CAL228F/CAL737R, ßt2a/ßt2b and CYLH3F/H3-1b. The sequences were deposited into GenBank (Accession Nos. for isolate SH161: OK326730 for ITS, OK413403 to OK413406 for TUB, CAL, HIS and TEF1-α; and isolate SH181: OK331347 for ITS, OK413407 to OK413410 for TUB, CAL, HIS, and TEF1-α). BLAST search of SH161 showed high similarities with sequences of Diaporthe eres (AR5193) [KJ210529 (ITS), Identities = 438/512, (94%); KJ420850 (HIS), Identities = 466/472, (99%); KJ210550 (TEF1-α), Identities = 345/350, (99%); KJ434999 (CAL), Identities = 344/345, (99%); KJ420799 (TUB), Identities = 508/517, (98%)]. BLAST results of SH181 are listed in Supplementary Table 1. Maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and MrBayes v. 3.2.6 with the concatenated sequences placed SH161 and SH181 in the clade of D. eres. Based on the multi-locus phylogeny and morphology, two isolates were identified as D. eres. The pathogenicity was tested on 1-yr-old cuttings of V. odoratissimum var. awabuki in the greenhouse. Healthy leaves were wounded with a sterile needle, then inoculated with 5-mm plugs from the edge of two isolates cultures. The PDA plugs were used for controls. Three plants were used for each treatment, and three leaves of each plant were inoculated. Each plant was covered with a plastic bag, and sterilized water was sprayed into the bags bidaily to maintain humidity and kept in a greenhouse at the day/night temperatures at 25 ± 2°C/16 ± 2°C. Three days after inoculation, the inoculated leaves appeared lesions similar to those in the field. The controls remained healthy. Diaporthe eres was reisolated from inoculated leaves. No fungus was isolated from controls. Diaporthe eres was reported from Viburnum lantana in Austria. Also, it was reported from V. odoratissimum and V. tinus in Ukraine. This is the first report of D. eres causing V. odoratissimum var. awabuki leaf spots in China. This finding will provide an effective basis for developing control strategies for the disease.

First Report of Diaporthe cercidis and D. nobilis Causing Leaf Blotch on Populus davidiana × P. bolleana in China.

Shanxin yang (Populus davidiana × P. bolleana) is a commercially valuable hybrid poplar that is widely planted in northern China. Efficient genetic transformation and gene-editing systems have been established for this hybrid poplar (Wang et al., 2011; Wang et al., 2020). However, records of fungal diseases on Shanxin yang are very limited. In July 2020, large necrotic lesions were observed on 16 one-year-old Shanxin yang seedlings planted in a greenhouse of Nanjing Forestry University, Nanjing, China. The disease symptoms appeared mostly on the leaves and not on the stems. Symptoms first manifested as differently sized and shaped brown spots, having clearly demarcated margins. As the disease progressed, the spots coalesced, and large lesions were present on the leaves. Severe infections resulted in whole-plant defoliation and eventually plant death. Small leaf samples (5 mm2) cut from lesion margins were surface sterilized with 75% ethanol for 30 s, followed by 1% NaClO for 90 s and then washed three times with sterile distilled water. After drying on sterilized filter paper, the cut tissues were plated on potato dextrose agar (PDA) supplemented with ampicillin (100 µg/mL) and incubated at 25°C in the dark. Three isolates (named as SX-1, SX-2 and SX-3, respectively) were obtained after 5 days. The isolation frequency was low, which might be due to the greenhouse microclimate that was unfavorable for pathogen spread. Mycelial plugs (5 mm) cut from the leading edge of the mycelia were transferred onto fresh PDA and synthetic nutrient-poor agar (SNA) plates to obtain pure cultures. On both media, colonies of the isolates were white on the front and light yellowish on the back, with concentric zonation. Abundant aerial mycelia developed; the hyphae were hyaline, non-septate, and approximately 0.794-2.961 µm in diameter. On the SNA medium, SX-1 and SX-3 produced globose to subglobose, black pycnidia after 18 days under a 12 h photoperiod. The alpha conidia were fusoid, aseptate, hyaline, smooth, and 6.4 ± 1.2 × 2.4 ± 0.6 µm (n = 50) in size. Under the same conditions, SX-2 produced pycnidia after 20 days, and the conidia were 2.8 ± 0.7 × 7.5 ± 1.3 µm. Beta conidia were not observed on any colony. Based on the morphological characteristics, the isolated mycelia resembled Diaporthe spp. (Gomes et al., 2013). To determine the species identity, genomic DNA from each isolate was extracted, and five loci were amplified, namely, part of the internal transcribed spacer (ITS) amplified with primers ITS1/ITS4 (White et al. 1990); part of the translation elongation factor 1-alpha (EF1-α) with primers EF1-728F/EF1-986R (Carbone and Kohn, 1999); part of the calmodulin (CAL) with primers CAL-228F/CAL-737R (Carbone and Kohn, 1999); part of the ß-tubulin (ß-tub) with primers Bt2a/Bt2b (Glass and Donaldson, 1995), and part of the histone H3 (HIS) with primers CYLH3F/H3-1b (Glass and Donaldson 1995, Crous et al., 2004). The obtained sequences were deposited in GenBank (accession numbers are listed in Table S1). BLAST analyses showed that the all the amplified fragments were highly homologous to Diaporthe spp. (Table S1). Based on concatenated sequences of the amplicons, a phylogenetic tree was constructed by using Maximum-likelihood and Bayesian inference methods. The multi-locus phylogenetic analyses distinguished SX-1 and SX-3 as D. cercidis, and SX-2 as D. nobilis. To complete Koch's postulates, the pathogenicity of SX-1, as well as SX-2, was tested on both detached and attached leaves of one-year-old Shanxin yang seedlings grown under greenhouse conditions. Healthy leaves were pierced with a sterile needle and then inoculated independently with 5-mm mycelial plugs cut from the edge of the 4-day-old colonies of SX-1 and SX-2 colonies. Controls were inoculated with noncolonized PDA plugs. Three replicates were prepared for each isolate. For the in-vitro tests, detached leaves were placed on wet filter paper in parafilm-sealed Petri dishes and cultured at 25 °C in the dark. For the attached leaf assays, the plants were kept in an 85% humidity chamber immediately after inoculation. All the inoculated leaves developed dark or brown necrotic lesions at 5 days after inoculation, whereas the control leaves showed no symptoms. D. cercidis and D. nobilis were separately reisolated from the inoculated leaves. The former was first described by Yang et al. (2018) as occurring on twigs and branches of Cercis chinensis, and very recently, this pathogen was reported to cause leaf blotch on Acer pictum subsp. mono (Wan et al., 2021). The latter infects some fruit trees (Yu et al., 2018; Sun et al., 2019; Ma et al., 2019) and chestnut (Zhang et al., 2018). All of these studies were conducted in China where there is a great diversity of Diaporthe species (Yang et al., 2018). To our knowledge, this is the first report of both D. cercidis and D. nobilis causing leaf blotch on poplar. The identification of these pathogens is essential for understanding the range of their host species and to manage the resulting fungal diseases, which may cause severe economic damage.

First Report of Leaf Rust on Blackberry (Rubus spp.) Caused by Kuehneola uredinis in Florida.

During the fall of 2020 and summer of 2021, symptoms of leaf rust were observed on blackberry plants of 'Kiowa', and breeding line 1734 (progeny of 'Natchez' and Arapaho') in a field trial at the University of Florida, Wimauma, FL. Symptoms consisted of small chlorotic spots (1 to 3 mm) on the upper side of the leaf, while the underside had yellow-orange pustules. Disease incidence was up to 100% on both 'Kiowa' and the breeding line 1734, and severity was up to 20% with most of the symptoms observed on older leaves. Two isolates were collected from 'Kiowa' and one from the breeding line 1734 for further investigation. Isolates were maintained and multiplied on healthy 'Kiowa' plants in growth chambers (25 ºC and 12-12 h photoperiod). Uredinia (n=30) were erumpent and ranged from 90 to 320 µm (Average=285 µm, SD=5.3 µm) in diameter. Urediniospores (n=50) were obovoid, yellow, and ranged from 24 to 36 µm long (Average=32 µm, SD=3.2 µm) and 22 to 30 µm wide (Average=28 µm, SD=2.5 µm). Based on morphology and literature, the pathogen was tentatively identified as Kuehneola uredinis (Link) Arth (Arthur 1906; Shands et al., 2018). Spores from a single uredinium of each isolate were collected with a needle and suspended in 50 µL of molecular biology-grade water yielding a final concentration of approximately 5 x 104 spores/mL. Two µL of each spore suspension was used for the PCR reactions. Two DNA fragments were amplified using the primers Rust2inv and LR6, and Rust18S-R and NS1 for the 5.8S-ITS2-28S gene region of rDNA (1,755 bp) and partial 18S gene region of rDNA (2,684 bp), respectively. The amplified products of the partial 28S gene region were sequenced with the primers LR3 and LR0R, and the 18S gene region with NS5, NS6, and NS4 (Aime 2006). DNA sequences were deposited in GenBank (accession nos. OK509845 - OK509848). BLASTn searches revealed that the isolates were 100% identical to K. uredinis reported causing leaf rust on blackberry in California (1044/1044bp, and 1540/1540bp for accession numbers MF158087, and MF158088, respectively). To test for pathogenicity, blackberry cultivars Kiowa, Natchez, Osage, Ouachita, Ponca, Prime-Ark® 45, Prime-Ark® Freedom, Prime-Ark® Traveler, and Prime-Ark® Horizon were inoculated. Five plants of each cultivar were inoculated with a mixture of spores of the three isolates, and two plants of each cultivar were used as controls. Spores were washed from leaves of 'Kiowa' exhibiting sporulation using a suspension of 1% Tween 20 in deionized water. The final concentration of the inoculum was adjusted to 104 spores/mL. Plants were inoculated in the greenhouse with a spray bottle until run-off and kept inside clear plastic boxes for 48 h. Controls were sprayed with sterile deionized water. Plants were watered by mists of 3 s every 10 min twice a week. Disease incidence and severity were evaluated weekly on five leaves per plant that had been tagged before inoculation. The experiment was repeated once. Symptoms identical to the original were only observed in 'Kiowa' and 'Prime-Ark® Freedom'. One week after inoculation, disease incidence was already 100% in both cultivars, with at least one pustule on all the inoculated leaves, and six weeks later disease severity was up to 50% (Average= 35%, SD=2.4%). To our knowledge, this is the first report of K. uredinis causing leaf rust on blackberry in Florida. This disease was reported on Rubus spp. in several U.S. states, and recently in California on Rubus ursinus (Farr and Rossman 2021; Shands et al. 2018). Blackberry is an emerging crop in Florida and efforts should be implemented to monitor the occurrence and spread of leaf rust considering that urediniospores disperse long distances by wind, especially if growers choose the susceptible cultivars 'Kiowa' and 'Prime-Ark Freedom'. The apparent resistance observed in other commercial cultivars such as 'Osage', 'Ouachita', and 'Ponca' may serve as valuable breeding parents for developing new blackberry cultivars with resistance to leaf rust.

First Report of Erysiphe magnoliicola Causing Powdery Mildew of Magnolia × soulangeana in China.

Magnolia × soulangeana Soul.-Bod., the saucer magnolia is an important woody ornamental plant cultivated widely in China, UK and USA. In August 2021, symptoms and signs of powdery mildew appeared on leaves of M. × soulangeana at the campus of Nanjing Forestry University (NJFU). The powdery mildew mainly infected young seedlings, with an incidence of 96.8% (436/450 seedlings), and some adult trees also been infected (5/30 trees). The mycelium was amphigenous, thinly effused or conspicuous, forming circular to irregular white patches. Noticeable brown lesions and necrosis occurred in the later stage of infection. Chasmothecia started to develop in October, 2021 and fully matured in early November, 2021. Ten fresh specimens were collected and examined to identify of the pathogen. Photos were taken with a ZEISS Axio Imager A2m microscope, a Zeiss stereo microscope (SteRo Discovery v20), and a scanning electronic microscope (JSM-7600F). Conidiophores arose from the upper part of mother cells, 78.5 ± 11.2× 10.9 ± 1.7 µm (n=30). Foot cells in conidiophores are straight and cylindrical with a constricted basal septum close to hyphal mother cell, 33.6 ± 4.3 × 10.3 ± 1.2 µm (n=30). Conidia were hyaline, ellipsoid to oval, solitary or in chains of two to four, 38.5 ± 3.3 × 18.4 ± 1.0 µm (n=30). Chasmothecia were amphigenous, scattered or aggregated, blackish brown, oblate, 101.1 ± 11.4µm diam. (n=30), with 6-10 appendages. Appendages were aseptate, rarely 1-septate, 5-6 times frequently dichotomously branched; tips were noticeably recurved, brown at the base, 105.1 ± 10.7 × 8.5 ± 1.4 µm (n=30). Asci were 6 to 8 per chasmothecium (n=30), ellipsoid to obovoid or saccate with a short stalk or sessile, 64.2 ± 6.5× 46.1 ± 5.7 um (n=30) in length, 4 to 6 spored. Ascospores were oblong-ovoid, 26.2 ± 1.4 × 13.8 ± 0.7 µm (n=30). Based on the morphological characteristics, the fungus was identified as Erysiphe magnoliicola S.E. Cho, S. Takam. & H.D. Shin. To confirm the causal fungus identity, a representative voucher specimen collected and deposited in herbarium of NJFU (NF50000008) was used for a phylogenetic analysis. Mycelia and conidia were collected from diseased leaves and genomic DNA of the pathogen was extracted. The internal transcribed spacer region (ITS) and large subunit (LSU) loci were amplified with primers ITS1/ITS4 and LR0R/LR05. The resulting sequences were deposited in GenBank (OL454094 for ITS, OM758416 for LSU). BLAST results showed that the ITS sequence was highly similar with a sequence of E. magnoliicola (type) [KJ567072, 614/619 (99.2%)], while LSU sequence was highly similar with E. magnoliicola [KJ567068, 889/891 (99.8%)] and E. magnoliae [JX235969, 903/909 (99.3%)]. Phylogenetic analyses using ITS and LSU sequences with maximum likelihood and Bayesian posterior probability using IQ-TREE v. 1.6.8 and MrBayes v. 3.2.6 placed this fungus in the E. magnoliicola clade. Based on the morphology and phylogeny, the fungus was identified as E. magnoliicola. Pathogenicity tests were carried out on six potted plants of M. × soulangeana. Three seedlings were inoculated by gently pressing the naturally infected leaves onto healthy leaves. Healthy leaves from three other seedlings served as control. Inoculated and control seedlings were placed in separate growth chambers at 23 ± 2°C/16 ± 2°C, 70% relative humidity, with a 16 h/8 h light/dark period. Symptoms developed 10 days after inoculation. The powdery mildew developing on the inoculated seedlings was examined, sequenced and confirmed as E. magnoliicola. The control leaves did not develop powdery mildew. Magnolia × soulangeana is a hybrid of Magnolia denudata × Magnolia liliiflora, both species, as well as M. sieboldii were already known as host plants of E. magnoliicola. This is the first report of powdery mildew caused by E. magnoliicola on M. × soulangeana. This finding provides crucial information for developing effective strategies to monitor and manage this disease.

Tracing the Origin and Evolutionary History of Pyricularia oryzae Infecting Maize and Barnyard Grass.

Blast disease is a notorious fungal disease leading to dramatic yield losses on major food crops such as rice and wheat. The causal agent, Pyricularia oryzae, encompasses different lineages, each having a different host range. Host shifts are suspected to have occurred in this species from Setaria spp. to rice and from Lolium spp. to wheat. The emergence of blast disease on maize in Iran was observed for the first time in the north of the country in 2012. We later identified blast disease in two additional regions of Iran: Gilan in 2013 and Golestan in 2016. Epidemics on the weed barnyard grass (Echinochloa spp.) were also observed in the same maize fields. Here, we showed that P. oryzae is the causal agent of this disease on both hosts. Pathogenicity assays in the greenhouse revealed that strains from maize can infect barnyard grass and conversely. However, genotyping with simple sequence repeat markers and comparative genomics showed that strains causing field epidemics on maize and on barnyard grass are different, although they belong to the same previously undescribed clade of P. oryzae. Phylogenetic analyses including these strains and a maize strain collected in Gabon in 1985 revealed two independent host-range expansion events from barnyard grass to maize. Comparative genomics between maize and barnyard grass strains revealed the presence or absence of five candidate genes associated with host specificity on maize, with the deletion of a small genomic region possibly responsible for adaptation to maize. This recent emergence of P. oryzae on maize provides a case study to understand host range expansion. Epidemics on maize raise concerns about potential yield losses on this crop in Iran and potential geographic expansion of the disease.

A new host record for Candidatus Phytoplasma cynodontis (16Sr XIV-A) associated with phyllody and fasciation of linseed (Linum usitatissimum) from India.

Linseed commonly called as flaxseed (Linum usitatissimum Linn.) is an important oilseed crop cultivated widely in Northern parts of Karnataka. During, 2019 (January-February), a characteristic disease was noticed with symptoms that resembled phytoplasma or like disease symptoms. The incidence was ranged from 6·5 to 16·5% in the experimental station of Raichur Agricultural University. The typical symptoms observed were virescence of floral parts, fasciation of the inflorescence axis, phyllody, stunted and flattened stem with reduced leaves. Symptomatic and healthy samples were collected and processed for molecular detection of phytoplasma. Total DNA was isolated from four infected plants and two healthy plants. The 16S rDNA region was amplified using P1/P7 followed by R16F2n/R16R2 primer pair which showed the amplification of expected amplicon size from all four infected samples. Furthermore, the SecA gene was amplified using SecA1/SecA3 primers. The PCR amplified products were subjected for direct sequencing from both directions and the consensus sequences were obtained and nBLAST search analysis revealed that the 16Sr RNA and SecA sequences were sharing maximum similarity (100%) with the reference sequence of Ca. P. cynodontis. The sequences were analysed phylogenetically by constructing a Phylogram independently by NJ method along with reference sequence of 16S rRNA region and SecA region retrieved from GenBank database showed that the phytoplasma sequence from linseed phyllody of the present study placed in a distinct clade along with reference sequence of "Ca. P. cynodontis" thus confirming the identity phylogenetically. Furthermore, iPhyClassifier and virtual RFLP proved that the phytoplasma belonged to 16SrXIV (subgroup A) phytoplasma. Previously linseed is known to be associated with 16SrII-D phytoplasma but the association of the 16SrXIV-A group of phytoplasma is not reported so far. Therefore, this is the new host record for Ca. P. cynodontis (16SrXIV-A) phytoplasma associated with linseed stem fasciation, phyllody from India.

First Report of Cercospora Leaf Spot Caused by Cercospora cf. flagellaris on Okra in China.

Okra [Abelmoschus esculentus (L.) Moench], which belongs to the family Malvaceae, is widely grown in the tropics, sub-tropics and warmer areas of the temperate zones for its immature seed pods which are consumed as a vegetable. In China, okra pods are consumed as not only vegetables but also as a traditional medicine to cure dental diseases and gastric ulcers. During September 2018 to June 2019, extensive spots on okra leaves were observed in several commercial fields (approximately 2.0 hectares), with disease incidence of approximately 25%~50% in the Yanqing District (115°98'E, 40°46'N) of Beijing, China. Symptoms of the disease initially appeared as small pale brown spots with yellow haloes. As the disease progressed, some spots gradually coalesced, forming larger irregular dark brown lesions. The centers of the lesions became grayish white. A total of 13 small fragments (3 to 5 mm) excised from the lesion margins were sterilized in 1% sodium hypochlorite (NaClO) for 1 min, followed by three washes with sterile distilled water, and then placed on potato dextrose agar (PDA) and incubated at 25°C in the dark for 5 days. In total, 21 cultures were obtained and purified by single-spore subcultures on PDA for morphological identification. The colonies on PDA were whitish to gray, with cottony aerial mycelium. Conidiophores were fasciculate, olivaceous brown, straight or geniculate, uniform in width, multiseptate, and ranged from 286/span> to 711 µm (avg. = 578 µm, n = 50). Conidia were hyaline, slightly curved or straight, needle shaped, truncate at the base, and terminal at the tip, 3-17-septate, and measuring 52 to 231 µm (avg. = 182 µm, n = 50). The morphological features were consistent with Cercospora cf. flagellaris Ellis & G. Martin (Groenewald et al. 2013). Pathogenicity tests were conducted on potted okra plants cv. 'Jiayuan'. Twenty four healthy okra plants at the true leaf stage were sprayed with conidial suspensions (1 × 106 conidia/mL), incubated at a glass cabinet maintained at 25°C and 90% relative humidity (RH). To each leaf approximately 10 mL of conidial suspension was applied. Plants sprayed with water were used as controls. Seven days later, dark brown spot, which were identical to those observed in the fields, were observed on inoculated leaves, whereas the control plants remained healthy. C. cf. flagellaris was reisolated from symptomatic leaves, confirming Koch's Postulates. Genomic DNA was extracted from fungal mycelium using the Plant Genomic DNA Kit (Tiangen Biotech Co. Ltd., Beijing, China). The nuclear ribosomal internal transcribed spacer region (ITS), and portions of the actin (ACT), histone H3 (HIS3), and translation elongation factor 1-α (TEF1) genes were amplified using primers ITS1/ITS4 (Groenewald et al. 2013), ACT-512F/ACT-783R (Carbone & Kohn 1999), CYLH3F/CYLH3R (Crous et al. 2006), and EF1-728F/EF1-986R (Carbone & Kohn 1999). The resulting 542 bp ITS, 226 bp ACT, 410 bp HIS3 and 306 bp TEF1 sequences of isolate QK14091813 were deposited in GeneBank (Accession nos. MT949700, MT949701, MT949702 and MT949703, respectively). The ITS, ACT, HIS3 and TEF1 sequences shared 99.42% to 100% identities to previously published sequences of C. cf. flagellaris (Accession nos. MN633275 for ITS, MF680960 for ACT, MK991295 for HIS3, and MK991292.1 for TEF1, respectively). Multi-locus phylogenetic analyses (ITS, ACT, HIS3, and TEF1) were performed by neighbor-joining method using MEGA 7.0. The resulting trees showed that C. cf. flagellaris isolate QK14091813 (this study) nested within the clade that includes other isolates of C. cf. flagellaris with a 99% confidence level. To our knowledge, this is the first report of C. cf. flagellaris causing leaf spot on okra (Farr and Rossman 2020). The pathogen has a worldwide distribution and an unusually broad host range, which can be of great significance, and the plant protection policy of priority to prevention and synthetical prevention should be followed.

Identification and Characterization of Nothophoma quercina Causing Bud Blight on Photinia × fraseri in China.

Photinia (Photinia × fraseri Dress) is a well-known green plant that has high ornamental value and is widely distributed around the world. An outbreak of typical bud blight disease was observed between May and August in photinia in 2017 in Qingdao, China. The causal agent for this blight was subsequently isolated from symptomatic samples and identified as Nothophoma quercina based on morphological characterization and molecular analyses (ITS, LSU, RPB2, and TUB2). Results of pathogenicity tests on isolated fungi also supported the conclusion that N. quercina is the pathogen responsible for this condition. To our knowledge, this is the first report of bud blight on P. fraseri caused by N. quercina in China.

First report of leaf spot caused by Colletotrichum siamense on Salix matsudana in China.

Salix matsudana Koidz. (Chinese willow) is an important landscaping tree species widely grown in China (Zhang et al. 2017). In October 2019, a characteristic leaf spot disease of S. matsudana was found on the campus of Nanjing Forestry University. Most 25-year-old S. matsudana trees (13 out of 21, approximately 62%) on campus showed the leaf spot disease. On average, 70% of the leaves per individual tree were affected by this disease. Foliar symptoms began as dark brown, irregular spots and the centers were gray-white, gradually enlarging with time. Leaf spot symptomatic leaves were collected from three infected S. matsudana trees (10 leaves/tree), and small infected tissues (3-4 mm2) were surface-sterilized in 75% ethanol for 30 s, 1% NaClO for 90 s, rinsed in ddH2O, dried on sterilized filter paper, and plated on potato dextrose agar (PDA), and then incubated at 25°C. Three isolates (NHY1-1, NHY1-2, and NHY1-3) of the same fungus were obtained in 85% of the samples and deposited in China's Forestry Culture Collection Center (NHY1-1: cfcc55354, NHY1-2: cfcc55355, NHY1-3: cfcc55359). The colonies of three isolates were white, but the reverse side was grayish-white. The conidia of NHY1-1 were one-celled, straight, subcylindrical, hyaline, 14.4 ± 0.9 × 5.4 ± 0.4 µm (n = 50), with a rounded end. Conidiophores were hyaline to pale brown, septate, and branched. Appressoria were one-celled, ellipsoidal, brown or dark brown, thick-walled, 8.0 ± 0.9 × 5.9 ± 0.5 µm (n = 50). The conidia and appressoria of the other two isolates weralmost identical to NHY1-1. The morphological characters of the three isolates were matched with those of the Colletotrichum gloeosporioides complex (Weir et al. 2012). For accurate identification, the DNA of the three isolates was extracted. The internal transcribed spacer region (ITS), actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), superoxide dismutase (SOD2), and ß-tubulin 2 (TUB2) genes were amplified using the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CL1C/CL2C, CHS-79F/CHS-345R, GDF1/GDR1, SODglo2-F/SODglo2-R, and Bt2a/Bt2b, respectively (Weir et al. 2012). The sequences were deposited in GenBank [Accession Nos. MW784679 and MW808959 to MW808964 for NHY1-1; MW784726 and MW808965 to MW808970 for NHY1-2; MW784729 and MW808971 to MW808976 for NHY1-3]. A BLAST search of GenBank showed that ITS, ACT, CAL, GAPDH, SOD2, and TUB2 sequences of the three isolates were identical to Colletotrichum siamense at a high level (>99%), and CHS-1 sequences of three isolates were consistent with Colletotrichum fructicola at a high level (>99%). A maximum likelihood and Bayesian posterior probability analyses using IQtree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (ITS, ACT, CAL, CHS-1, GAPDH, SOD2, and TUB2) placed NHY1-1, NHY1-2, and NHY1-3 in the clade of C. siamense with high bootstrap support values (ML/BI = 93/1). The pathogenicity of three isolates were tested on potted 2-yr-old seedlings (50-cm tall) of S. matsudana, which were grown in a greenhouse. Healthy leaves were wounded with a sterile needle and then inoculated with 10 µL of conidial suspension (106 conidia/mL). Controls were treated with ddH2O (Zhu et al. 2019). In total, 12 seedlings were inoculated including controls. Three seedlings/isolate and 10 leaves/seedling were used for each treatment. The plants were covered with plastic bags after inoculation and sterilized H2O was sprayed into the bags twice/day to maintain humidity and kept in a greenhouse at the day/night temperatures at 25 ± 2 / 16 ± 2°C. Within 7 days, all the inoculated points showed lesions similar to those observed in field, whereas controls were asymptomatic. The infection rate of each of the three isolates is 100%. C. siamense was re-isolated from the lesions, whereas no fungus was isolated from control leaves. The diseases caused by C. siamense often occur in tropical and subtropical regions of China, with a wide range of hosts, such as Hevea brasiliensis and Coffea arabica, etc. (Cao et al. 2019; Liu et al. 2018). This is the first report of C. siamense causing leaf spot of S. matsudana in China and the world. These data will help to develop effective strategies for managing this newly emerging disease.