Neofusicoccum cryptomeriae sp. nov. and N. parvum Cause Stem Basal Canker of Cryptomeria japonica in China.

Cryptomeria japonica D. Don is a coniferous tree species widely grown in southern China for its high ornamental value. Recently, during disease surveys in China, a symptom of dieback occurred on C. japonica in Nanjing, Jiangsu Province, China. A total of 130 trees were surveyed and more than 90% showed the same symptom. The crowns of affected trees were brown when viewing from a distance, and the bark showed no difference from the healthy ones. In this study, 157 isolates were isolated from the 3 affected plants of C. japonica, and based on the living culture on PDA, the fungal isolates were preliminarily divided into 6 groups. Thirteen representative isolates were selected for the pathogenicity test, and seven of them showed obvious pathogenicity on C. japonica, causing stem basal canker. These isolates were identified based on comparisons of the DNA sequences of the internal transcribed spacer regions (ITS), partial translation elongation factor 1-alpha (tef1), ß-tubulin (tub2), and DNA-directed RNA polymerase II subunit (rpb2) and combined with their morphological characteristics. Results showed that these seven isolates belong to two taxa in Neofusicoccum, including a species new to science. The new species, Neofusicoccum cryptomeriae, was hereby described and illustrated. The other species was N. parvum. Both species were pathogens of stem basal canker of Cryptomeria japonica.

Three New Species of Diaporthe Causing Leaf Blight on Acer palmatum in China.

Diaporthe spp. are often reported as plant pathogens, endophytes, and saprobes. In this study, three new species (Diaporthe foliicola, D. monospora, and D. nanjingensis) on Acer palmatum were described and illustrated based on morphological characteristics and phylogenetic analyses. Phylogenetic relationships of the new species were determined by multilocus phylogenetic analyses based on partial sequences of the internal transcribed spacer (ITS) region, translation elongation factor 1-α (TEF), ß-tubulin (TUB), histone H3 (HIS), and calmodulin (CAL) genes. Genealogical concordance phylogenetic species recognition with a pairwise homoplasy index test was used to verify the conclusions of the phylogenetic analyses. All species were illustrated and their morphology and phylogenetic relationships with other related Diaporthe spp. are discussed. In addition, the tests of Koch's postulates showed that the three new species were pathogens causing leaf blight on A. palmatum.

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.

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 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.

First Report of Diaporthe fusicola Causing Leaf Blotch of Osmanthus fragrans in China.

Osmanthus fragrans Lour. is widely distributed in China, Japan, Thailand and India (Zang et al., 2003) and one of the top 10 most well-known flowering plants in China. Since February, 2017, a foliar disease, with a disease incidence of ~60%, occurred on O. fragrans in a community park in Luzhai, Guangxi, China. Symptoms began as round or irregular small yellow spots and became pale brown to gray-brown with time. Small leaf tissues (3 to 4 mm2) cut from lesion margins were surface-sterilized in 75% ethanol for 30 s and 1% NaClO for 90 s before they were rinsed in ddH2O and dried on sterilized filter paper. After drying, the sterilized tissues were plated on potato dextrose agar (PDA) and incubated at 25°C in the dark for 5 days. Five single-spore isolates were obtained and a representative isolate (GH3) was selected and deposited in the China's Forestry Culture Collection Center. The colony on PDA was white with concentric zonation and white aerial mycelia, but the reverse was yellow. Black pycnidia developed on alfalfa extract + Czapek at 25°C with a 14/10 h light/dark cycle after 17 days. Conidiophores were hyaline, branched, septate, straight to sinuous, 12.4-24 × 1.9-2.5 µm (n = 20). The conidia were fusoid, hyaline, smooth, mostly 2-guttules and measured 7.2 ± 0.7 × 2.3 ± 0.2 µm (n = 50). The morphological characters of pycnidia, conidiophores and conidia of all five isolates matched those of Diaporthe spp. (Gomes et al. 2013). DNA of isolates GH3, GH7 and GH8 was extracted and the internal transcribed spacer region (ITS), partial sequences of elongation factor 1-alpha (EF1-α), calmodulin (CAL), beta-tubulin (ß-tub) and histone H3 (HIS) genes were amplified with primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R and CAL228F/CAL737R (Carbone et al. 1999), ßt2a/ßt2b and CYLH3F/H3-1b (Glass and Donaldson 1995, Crous et al. 2004), respectively. The sequences of GH3, GH7 and GH8 were deposited in GenBank (GH3: Accession nos. MT499213 for ITS, MT506473 to MT506476 for EF1-α, ß-tub, HIS, and CAL; GH7: MT856374 and MT860397 to MT860400; GH8: MT856375 and MT860401 to MT860404). BLAST results showed that the ITS, EF1-α, ß-tub, HIS, and CAL sequences of GH3 were highly similar with sequences of Phomopsis sp. [LC168784 (ITS), Identities = 506/506(100%)], Diaporthe fusicola [MK654863 (EF1-α), Identities = 274/275(99%)], D. amygdali [MK570513 (ß-tub), Identities = 461/461(100%)], D. fusicola [MK726253 (HIS), Identities = 403/403(100%)] and D. amygdali [KC343263 (CAL), Identities = 428/428(100%)], respectively. 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 isolates GH3, GH7 and GH8 in the D. fusicola cluster and separated them from D. eres and D. osmanthi, which were previously reported from Osmanthus spp. (Gomes et al., 2013; Long et al., 2019). Based on the multi-gene phylogeny and morphology, all three isolates were identified as D. fusicola. The pathogenicity of GH3 was tested on 1-yr-old seedlings of O. fragrans. Healthy leaves were wounded with a sterile needle and then inoculated with either 5-mm mycelial plugs cut from the edge of a 5-day-old culture of GH3 or 10 µL of conidial suspensions (106 conidia/mL). Control leaves were treated with PDA plugs or ddH2O. Three plants were used for each treatment. The plants were covered with a plastic bag 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°C/16 ± 2°C. Lesions appeared 3 days later. No lesions were observed on control leaves. The same fungus was re-isolated from lesions. This is the first report of D. fusicola causing leaf blotch on O. fragrans. These results form the basis for developing effective strategies for monitoring and managing this potential high-risk disease.

First Report of Leaf Blotch of Salix babylonica Caused by Botryosphaeria dothidea in China.

Salix babylonica L. (weeping willow) is an important ornamental tree commonly planted in China. Since 2018, a new disease with a high incidence has been observed on S. babylonica at the campus of Nanjing Forestry University (NFU), Nanjing, Jiangsu, China. The symptoms began as small dark brown lesions formed along the leaf margins and tips; and later became gray to brown in the center with dark brown borders. Small samples (3 to 4 mm2) from the lesion margins were surface-sterilized with 75% ethanol for 30 s and 1% NaClO for 90 s. Subsequently samples were, rinsed with sterile H2O, plated on potato dextrose agar (PDA) and incubated at 25°C. The same fungus was isolated in 95% of the samples. Pure cultures were obtained by monosporic isolation. A representative isolate, NFS1 was used for morphological and molecular characterization and deposited in China's Forestry Culture Collection Center (cfcc 54212). On PDA, colonies were initially white and gradually became grayish-green to dark gray from the center to the edge. After 1 week, colonies turned dark, and after 3 weeks black pycnidia developed on the surface of media. Conidia were one-celled, hyaline, smooth, and fusoid to ellipsoid. Conidia measurements were 23.0 ± 1.9 × 5.8 ± 0.7 µm (n = 50). The morphology matched the description of Botryosphaeria dothidea (Slippers et al. 2004). For an accurate identification, genomic DNA of NSF1 was extracted to amplify the internal transcribed spacer (ITS) region, the transcription eongation factor (tefa-1), beta-tubulin (ß-tub), the large subunit (LSU), and small subunit (SSU) genes with the specific primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn, 1999), ßt2a/ßt2b (Glass and Donaldson, 1995), LR0R/LR05 (Schoch et al. 2009), and NS1/NS4 (White et al. 1990), respectively. The sequences were deposited in GenBank (Accession Nos. MN826233 for ITS, MN855215 for tefa-1, MN855216 for ß-tub, MN886965 for LSU, and MN886966 for SSU). A BLAST search of GenBank showed that the ITS, tefa-1, ß-tub, LSU and SSU sequences of NSF1 were similar to those of B. dothidea KY788304 (Identity = 527/532; 99%), MG459974 (Identity = 247/247; 100%), MH724212 (Identity = 404/404; 100%), DQ377850 (Identity = 865/867; 99%), and KX091154 (Identity = 1,043/1,045; 99%), respectively. A maximum likelihood and Bayesian posterior probability-based phylogenetic analyses using IQ-tree v. 1.6.8 and Mr. Bayes v. 3.2.6 with the concatenated sequences (ITS, tefa-1, ß-tub, LSU, and SSU) placed NFS1 in the clade of B. dothidea. Based on the multi-gene phylogeny and morphology, NFS1 isolate was identified as B. dothidea. To fulfill Koch's postulates, 20 detached and 20 attached healthy 10-week-old leaves from three 30-year-old S. babylonica plants at the campus of NFU were inoculated with 5-mm mycelial plugs of isolate NFS1 of 3-day-old cultures grown on PDA. Control leaves were treated with agar plugs. The detached inoculated leaves were placed in Petri dishes on a piece of wet filter paper and incubated at 25°C. The attached leaves were enclosed in a plastic bag along with the branches with a wet cotton ball inside. Sterile H2O was sprayed into the plastic bags twice daily to keep moisture conditions and incubated for 5 days. The experiment was repeated two times. Within 5 days, all the inoculated points showed lesions similar to those obsrved in the field, whereas controls were asymptomatic. The same fungus was re-isolated from these lesions with a frequency of 100%. B. dothidea has been reported to infect a broad range of hosts, including S. babylonica in the USA (Grand 1985). This is the first report of B. dothidea on S. babylonica in China. This finding provides crucial information on this high risk disease to willow and basis for identifying management strategies.