First Report of Leaf Spot on Cherry Tomatoes Caused by Exserohilum rostratum in Hainan Province, China.

Cherry tomatoes (Lycopersicon esculentum var. cerasiforme) is the main tomato variety planted in Hainan Province, China and is prized for its nutritional value and sweet taste (Zheng et al. 2020). During October 2020 to February 2021, a leaf spot disease was observed on cherry tomatoes (cultivar Qianxi) in Chengmai, Hainan Province. The disease incidence was approximately 40% in each of three fields in Yongfa (19°76'-21°08'N, 110°21'-110°51'E). Leaves were initially chlorotic before developing black, irregular-shaped lesions on the leaf margins or tips. After several days, lesions expanded along the mid-vein to encompass the entire leaf. Then, the affected leaves turned gray-brown, leading to defoliation. Severely affected leaves became dry and necrotic. Leaf tissues of 10 diseased plants samples collected from the fields were surface sterilized in 70% ethanol for 30 s, 0.1% HgCl2 for 30 s, rinsed thrice with sterile distilled water for 30 s, placed on a modified potato dextrose agar (PDA) with 30 mg/liter of kanamycin sulfate, and incubated at 28°C in the dark for 3 to 5 days. Three fungal isolates were obtained from the diseased leaves by single-sporing. The mycelia on PDA were white and later became gray or dark gray after 3 to 4 days. Conidia were rostrate, straight to slightly curved, ellipsoidal to narrowly obclavate, dark brown, protuberant with a darker and thicker wall at the basal end. Conidia were 4 to 12 distoseptate and measured 63.92 ± 5.77 × 13.47 ± 1.22 µm (n= 50) Conidiophores were single, cylindrical, dark brown, geniculate, with swollen conidiogenous cells containing a acircular conidial scar. Morphological characteristics of the isolates were similar to those of Exserohilum rostratum (Cardona et al. 2008). A representative isolate (FQY-7) was used for pathogenicity and genomic studies. Genomic DNA was extracted from the mycelium of a representative isolate (FQY-7). The internal transcribed spacer (ITS) region, actin (act), translation elongation factor 1-alpha (tef1-α), glyceraldehydes 3-phos-phate dehydrogenase (gapdh) and ß-tubulin (tub2) genes were amplified with primers ITS1/ITS4 (White et al. 1990), Act1/Act4 (Voigt and Wöstemeyer 2000), EF1-728F/EF1-986R (Carbone and Kohn 1999), Gpd-1/Gpd-2 (Berbee et al. 1999) and T1 (O'Donnell and Cigelnik 1997) + Bt2b (Glass and Donaldson 1995). The consensus sequences (GenBank Accession No. MW036279 for ITS, MW133266 for act, MW133268 for tef1-α, MW133267 for gapdh, and MW133269 for tub2) were aligned using BLAST in GenBank obtaining 100%, 100%, 99%, 100%, and 99% identity to E. rostratum strain CBS706 (LT837842, LT837674, LT896663, LT882546, LT899350). Maximum likelihood analysis based on the combined five gene sequences was conducted under 1,000 bootstrap replicates. The Phylogenetic tree showed that FQY-7 and E. rostratum were located in one clade supported with 99% bootstrap values. Pathogenicity test was performed by depositing 10-µl droplets of a conidial suspension (1 × 106 per ml) into 5 noninoculated leaves (using a sterile needle) of 10 healthy 5-month-old cherry tomato (cv. Qianxi) plants. An equal number of artificially control leaves were received only sterile water to serve as a negative control. The test was conducted three times. Plants were kept at 28°C with 80% humidity and observed for symptoms every day. Two weeks after inoculation, all the inoculated plants showed symptoms of black spots similar to those observed in the field. No symptoms were observed on the controls. FQY-7 was successfully re-isolated from the inoculated leaves and confirmed by morphological characterization and molecular assays as described herein. To the best of our knowledge, this is the first report of leaf spot of cherry tomatoes caused by E. rostratum in China. Confirming the existence of this pathogen in this area will be useful to adopt effective field management measures to control this disease on cherry tomatoes. References: Berbee, M. L., et al. 1999. Mycologia 91:964. Cardona, R. et al. 2008. Bioagro 20:141. Carbone, I. and Kohn, L. M. 1999. Mycologia 91:553. Glass, N. L., and Donaldson, G. C. 1995. Appl. Environl. Microb. 61:1323. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. O'Donnell K., and Cigelnik, E. 1997. Mol. Phylogenet. Evol. 7:103. Voigt, K., and Wöstemeyer, J. 2000. Microbiol. Res. J. 155:179. Zheng J., et al. 2020. Guangdong Agr. Sci. 47:212. The author(s) declare no conflict of interest.

First Report of Diaporthe longicolla Causing Leaf Spot on Kalanchoe pinnata in China.

Kalanchoe pinnata (Lam.) Pers. [syn.: Bryophyllum pinnatum (Lam.) Oken] is an important medicinal agent in southern China. The succulent leaves of this plant are used in the treatment of cholera, bruises, uri-nary diseases and whitlow. In Oct. 2019, leaf spots were detected on K. pinnata plants in Chengmai County, Hainan Province, China. Lesions with brown to black margins were irregularly shaped and associated with leaf margins. Spots coalesced to form larger lesions (Fig. S1-A), with black pycnidia present in more mature lesions. Symptomatic K. pinnata were found with 10-20% incidence during the humid winters of Hainan Province. Leaf tissues of 10 symptomatic plants were collected and surface sterilized in 70% ETOH for 30s, 0.1% HgCl2 for 30 s, rinsed 3x with sterile distilled water for 30s, placed on potato dextrose agar (PDA) amended with 30mg/L of kanamycin sulfate, and incubated at 25°C in the dark for 3-5 days. Four fungal isolates were obtained using a single-spore isolation method. The colonies were floccose, dense, and white with forming on older colonies grown on PDA (Fig. S1-B-1&2). Alpha conidia exuded from ostiole, rostrate, long-beaked pycnidia in creamy-to-yellowish drops. Alpha conidia were hyaline, ellipsoidal, separated and averaged 6.3µm (SD ± 1.13) long × 1.9µm (SD ± 0.33) wide (n=50). Beta conidia were not seen. The morphological characteristics matched the previous description of Diaporthe longicolla (syn. Phomopsis longicolla) (Hobbs et al. 1985). Mycelial genomic DNA of the representative isolate LDSG3-2 was extracted as template. The internal transcribed spacer (ITS) , translation elongation factor 1α gene (TEF) and ß-tubulin (TUB2) regions were amplified. These loci were amplified using primer pairs ITS4/ITS5 (White, et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999) and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. A BLAST search of GenBank showed ITS (MN960195), TEF (MN974483) and TUB2 (MN974482) sequences of the isolate were 99%, 100%, and 99% homologous with D. longicolla strains DL11 (MF125048, 557/563 bp), D55 (MN584792, 347/347 bp) and DPC-HOH-32 (MK161506, 502/504 bp). Maximum likelihood trees based on concatenated nucleotide sequences of the three genes were constructed using MEGA 7.0, and bootstrap values indicated the isolate was D. longicolla (Fig. S1-D). Pathogenicity testing was performed using isolate LDSG3-2 by depositing 5µl droplets of a conidial suspension (1 × 106 ml-1) into 5 artificially wounded leaves (using a sterile needle) of 10 healthy 3-month-old K. pinnata plants. An equal number of artificially wounded control leaves were inoculated with sterile water to serve as a negative control. The test was conducted three times. Plants were kept at 25°C in 80% relative humidity and observed for symptoms. Two weeks after inoculation, no symptoms were observed on control plants (Fig. S1-C-1) and all inoculated plants showed symptoms (Fig. S1-C-2) similar to those observed in the field. The fungus was re-isolated from the infected tissues and showed the same cultural and morphological characteristics of the strain inoculated and could not be isolated from the controls fulfilling Koch's postulates. To our knowledge, this is the first report of leaf spot on K. pinnata caused by D. longicolla in China. This disease is of concern since Phomopsis diseases are common in K. pinnata fields and can cause significant reduction in yield. References: White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. DOI: 10.1016/0167-7799(90)90215-J Carbone, I., and Kohn, L. M. 1999. Mycologia. 91:553. DOI: 10.2307/3761358 Glass, N. L., and Donaldson, G. C. 1995. Appl. Environ. Microbiol. 61:1323. DOI: 10.1002/bit.260460112 Hobbs, T. W. et al. 1985. Mycologia. 77: 535. DOI: 10.2307/3793352.

First Report of Colletotrichum gloeosporioides Causing Anthracnose on Canna edulis Ker in China.

Canna edulis Ker has been an important economic plant in southern China. The tuberous stems are the most valued plant part and in the past were routinely used as animal feeds. In June 2019, leaf spot disease were detected on C. edulis plants in Chengmai, Hainan Province. Symptoms of the disease were characterized by oval-shaped, initially pale to yellow lesions that become necrotic (brown) with yellow borders, As the lesions expanded, the disease could encompass the entire leaves. which were seen as concentric rings typical of anthracnose disease (FigS1-A). A survey of C. edulis plants revealed that the disease caused serious damage during the summer in Hainan Province, with 50 to 60% incidence in plants. To isolate the pathogen, ten pieces of diseased leaf samples were plated and seven pieces yielded fungal colonies after 5 to 6 days of incubation at 25 °C. The Colonies were single-spored to obtain pure cultures. Pure cultures on potato dextrose agar (PDA) appear white to gray, with white margins and aerial hyphae, and the reverse of the colonies was gray to brown (FigS1-B). Conidia were single-celled, hyaline, cylindrical to slightly curved with a rounded apex and truncated base that measured 13.3 to 18.1(length) × 3.7 to 5.5 (width) µm (n=50) (FigS1-C). The morphological characteristics and measurements of this fungal pathogen matched the previous descriptions of Colletotrichum gloeosporioides (Prihastuti et al. 2012). Isolate JO-3 was identicated by molecular analysis, sequences of the internal transcribed spacer (ITS), actin (ACT) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) regions were obtained. These loci were amplified from isolates using the primer pairs ITS1/ITS4 (Mills et al. 1992), ACT-512F/ ACT-783R (Weir et al. 2012) and GDF/GDR (Templeton et al. 1992) respectively. A BLAST search of GenBank showed that the ITS (MN913584), ACT(MN919196) and GAPDH (MN919195) sequences of the isolate were 99% , 100% and 100% homologous with C. gloeosporioides (GenBank accession nos. MH930419, JX009931 and KX885158). Maximum likelihood trees based on concatenated sequences of the three genes were constructed using MEGA7.0. The results showed the strains isolated from C. edulis were closely related to C. gloeosporioides, as supported by high bootstrap values (FigS1-D). Pathogenicity test was performed with isolate JO-3 by depositing 10-µl droplets of a suspension (1 × 106 conidia/ml) on the surfaces of five artificially wounded leaves (a small hole made with a pushpin) of ten healthy 1-year-old C. edulis plants. An equal number of control leaves were inoculated with sterile water to serve as a negative control. The test was conducted three times. Plants were kept at 25°C with 80% humidity and observed for symptom every day. One weeks after inoculation, all the inoculated plants showed symptoms of yellow sunken spots similar to those observed in the field. No symptoms were observed on the controls. The fungus re-isolated from the infected tissues showed the same cultural and morphological characteristics of the strain inoculated, fulfilling Koch's postulates. C. gloeosporioides was previously reported as the causal agent of anthracnose on Hymenocallis littoralis (Zhao et al. 2019), Abelmoschus esculentus (L.) Moench (Shi et al. 2019) and Sorbaria sorbifolia (Li et al. 2019) in China. To our knowledge, this is the first report of anthracnose on C. edulis caused by C. gloeosporioides in China. This disease can seriously affect the yield and quality of C. edulis in China. Given its wide host range, C. gloeosporioides has great potential to become an economically important plant pathogen. The project was partially funded by Hainan Provincial Research Institute of technology development projects (Collection, Evaluation and Domestication Cultivation of Wild Vegetable Germplasm Resources in Hainan), Hainan Provincial Key Laboratory for Vegetables and Biology，Hainan Provincial Engineering Research Center for Melon and Vegetable Breeding, Major scientific and technological projects in Hainan Province(ZDKJ2017001)，Key R & D projects in Hainan Province (ZDYF2019066), The third Survey and Collection of Crop Germplasm Resources in China. References: Mills, P. R., et al. 1992. FEMS Microbiol Lett. 98:137-144 Weir, B. S., et al. 2012. Stud. Mycol. 73:115. Templeton, M.D. et al. 1992. Gene. 122:225. Prihastuti, H., et al. 2009. Fungal Divers. 39:89 C. D. Zhao, et al. 2019. Plant Dis.103:3286 Y. X. Shi, et al. 2019. Plant Dis.103:303 X. Y. Li, et al. 2019. Plant Dis.103:242.

First Record of Leaf Spot Disease on Costus speciosus Caused by Nigrospora oryzae in Hainan, China.

Costus speciosus (Koen.) Smith has been an important medicinal agent in the various traditional and folk systems of medicine in southern China. In September 2018, leaf spot disease was detected on C. speciosus plants in Chengmai County, Hainan Province. A survey of C. speciosus plants revealed that the disease caused serious damage during the typhoon season of September to November in Hainan Province, with 80 to85% incidence in plants. Early symptoms were yellow-to-brown, irregular-shaped lesions on the leaf margin or tip. After several days, lesions expanded along the mid-vein until the entire leaf was destroyed. Then, the infected leaves turned gray brown, leading to defoliation. Heavily infected leaves became dry and died. The pathogen was consistently isolated from the lesions and pure single-spore cultures were obtained. Twenty pieces of diseased leaf samples were plated and seven pieces yielded fungal colonies after 5 to 6 days of incubation at 25 °C. Colonies on potato dextrose agar (PDA) were white and later became gray to black. Conidia were unicellular, terminal, black, elliptical that measured 10 to 13 (length) × 12 to 16 (width) µm (n=30), growing aerial mycelium covering the entire petri dish (9 cm in diameter). The morphological characteristics and measurements of this fungal pathogen matched the previous descriptions of Nigrospora oryzae (Wang et al. 2017). To confirm identity the internal transcribed spacer (ITS) region of the ribosomal DNA was amplified using primers ITS1/ITS4 (Mills, P. R., et al. 1992), and the 530-bp product (GenBank Accession No. MK280694) of the ITS showed 99% sequence identity to N. oryzae isolates TLFa21 (GenBank Accession No. MH790146) and xsd08022 (GenBank Accession No. EU918714). Pathogenicity tests were conducted. Three leaves of three C. speciosus plants were wounded and inoculated with mycelial plugs (5×5mm) , and an additional five plants were inoculated with PDA plugs as a control. All plants were placed in the field and temperature ranged from 23 to 29°C. Ten days after inoculation, all the inoculated plants showed typical leaf spot symptoms, a yellow-to-brown mildew appeared at the points of inoculation. No symptoms were observed on the controls. The fungus was re-isolated from the infected tissues, fulfilling Koch's postulates. N. oryzae was previously reported as the causal agent of leaf spot on cotton (Zhang. et al. 2012), dendrobium candidum (Wu. et al. 2014) and Aloe vera (Zhai. et al. 2013) in china. To our knowledge, this is the first report of leaf spot of C. speciosus caused by N. oryzae in China. The project was partially founded by Hainan Provincial Research Institute of technology development projects (Screening and application of endophytic bacteria with high resistance to Fusarium Wilt of Sauropus androgynus), Hainan Provincial Key Laboratory for Vegetables and Biology，Hainan Provincial Engineering Research Center for Melon and Vegetable Breeding, Major scientific and technological projects in Hainan Province(ZDKJ2017001)，Third Survey and Collection of Crop Germplasm Resources in China, Collection, identification and preservation of pathogenic bacteria of inverted season vegetable in Hainan. References: L. F. Zhai., et al.2013. Plant Dis.97:1256 L. X. Zhang., et al.2012. Plant Dis.102:2029 J. B. Wu., et al.2014. Plant Dis.98:996 Mills, P. R., et al. 1992. FEMS Microbiol Lett. 98:137-144 Wang et al. 2017. Persoonia 39: 118-142.