First Report of Bipolaris oryzae Causing Leaf Spot on Cultivated Wild Rice (Oryza rufipogon) in China.

Wild rice (Oryza rufipogon) has been widely studied and cultivated in China in recent years due to its antioxidant activities and health-promoting effects. In December 2018, leaf spot disease on wild rice (O. rufipogon cv. Haihong-12) was observed in Zhanjiang (20.93 N, 109.79 E), China. The early symptom was small purple-brown lesions on the leaves. Then, the once-localized lesions coalesced into a larger lesion with a tan to brown necrotic center surrounded by a chlorotic halo. The diseased leaves eventually died. Disease incidence was higher than 30%. Twenty diseased leaves were collected from the fields. The margin of diseased tissues was cut into 2 × 2 mm2 pieces, surface-disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s, and then rinsed three times with sterile water before isolation. The tissues were plated on potato dextrose agar (PDA) medium and incubated at 28 °C in the dark for 4 days. Pure cultures were produced by transferring hyphal tips to new PDA plates. Fifteen isolates were obtained. Two isolates (OrL-1 and OrL-2) were subjected to further morphological and molecular studies. The colonies of OrL-1 and OrL-1 on PDA were initially light gray, but it became dark gray with age. Conidiophores were single, straight to flexuous, multiseptate, and brown. Conidia were oblong, slightly curved, and light brown with four to nine septa, and measured 35.2-120.3 µm × 10.3-22.5 µm (n = 30). The morphological characteristics of OrL-1 and OrL-2 were consistent with the description on Bipolaris oryzae (Breda de Haan) Shoemaker (Manamgoda et al. 2014). The ITS region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor (EF-1α) were amplified using primers ITS1/ITS4, GDF1gpp1/GDR1 gdp2 (Berbee et al. 1999), and EF-1α-F/EF-1α-R EF-1/EF-2 (O'Donnell 2000), respectively. Amplicons of OrL-1 and OrL-2 were sequenced and submitted to GenBank (accession nos. MN880261 and MN880262, MT027091 and MT027092, and MT027093 and MT027094). The sequences of the two isolates were 99.83%-100% identical to that of B. oryzae (accession nos. MF490854，MF490831，MF490810) in accordance with BLAST analysis. A phylogenetic tree was generated on the basis of concatenated data from the sequences of ITS, GAPDH, and EF-1α via Maximum Likelihood method, which clustered OrL-1 and OrL-2 with B. oryzae. The two isolates were determined as B. oryzae by combining morphological and molecular characteristics. Pathogenicity test was performed on OrL-1 in a greenhouse at 24 °C to 30 °C with 80% relative humidity. Rice (cv. Haihong-12) with 3 leaves was grown in 10 pots, with approximately 50 plants per pot. Five pots were inoculated by spraying a spore suspension (105 spores/mL) onto leaves until runoff occurred, and five pots were sprayed with sterile water and used as controls. The test was conducted three times. Disease symptoms were observed on leaves after 10 days, but the controls remained healthy. The morphological characteristics and ITS sequences of the fungal isolates re-isolated from the diseased leaves were identical to those of B. oryzae. B. oryzae has been confirmed to cause leaf spot on Oryza sativa (Barnwal et al. 2013), but as an endophyte has been reported in O. rufipogon (Wang et al. 2015).. Thus, this study is the first report of B. oryzae causing leaf spot in O. rufipogon in China. This disease has become a risk for cultivated wild rice with the expansion of cultivation areas. Thus, vigilance is required.

First Report of Leaf Spots caused by Nigrospora oryzae on Wild Rice in China.

In recent years, wild rice (Oryza rufipogon Griff) has been widely cultivated because of its health-promoting effects. In May 2019, leaf spot lesions on cv. Haihong-12 were observed in Zhanjiang (20.93N, 109.79E), China. Leaf symptoms were yellow-to-brown, oval or circular with a very distinctive, large yellow halo. Black spores appeared on the leaves with advanced symptoms. The lesions coalesced, causing the entire leaf to become blighted and die. Disease incidence reached approximately 10% in the fields (8 ha) surveyed. Twenty leaves with symptoms were collected and cut into pieces of 2 ×2 cm in size. They were surface-disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite (NaOCl) for 60 s, rinsed three times with sterile water, blotted dry on sterile paper, plated on potato dextrose agar (PDA) medium, and incubated at 28°C in the dark for 4 days. Ten pure cultures were obtained by transferring hyphal tips to new PDA plates, and monosporic cultures were obtained from three isolates (Nos-1, Nos-2, and Nos-3). Those isolates exhibited very similar morphological characteristics on PDA. Colony of isolate Nos-1 was white at the early stage and became dark gray after 7 days. Conidia were produced from clusters of conidiophores, single celled, black, smooth, spherical, and 9.5 to 14.2 µm (average 10.6 µm ± 0.42) in diameter. Morphological characteristics of the isolates matched the description of Nigrospora oryzae Petch (Wang et al. 2017). The ITS region was amplified using primers ITS1 and ITS4 (White et al. 1990). Nucleotide sequences of isolates Nos-1, Nos-2, and Nos-3 deposited in GenBank under acc. nos. MW042173, MW042174, and MW042175, respectively, were 100% identical to N. oryzae (acc. nos. KX985944, KX985962; and KX986007). A phylogenetic tree generated based on the ITS sequences and using a Maximum Likelihood method with 1,000 bootstraps showed that these three isolates from wild rice were grouped with other N. oryzae isolates downloaded from GenBank (bootstrap = 100%) but away from other Nigrospora spp. Pathogenicity test was performed with these three isolates in a greenhouse at 24 to 30°C. Approximately 50 seedling of wild rice cv. Haihong-12 were grown in each pot. At the 3-leaf stage, plants in three pots were inoculated with each isolate by spraying a spore suspension (105 spores/ml) until runoff. Three pots sprayed with sterile water served as the controls. Each 3-pot treatment was separately covered with a plastic bag. The test was conducted three times. Diseased symptoms were observed on the inoculated leaves after 10 days while no disease was observed in the control plants. Morphological characteristics and the ITS sequences of fungal isolates re-isolated from the diseased leaves were identical to those of N. oryzae. N. oryzae has been reported to cause leaf spot on O. sativa (Wang et al. 2017), but not on O. rufipogon. Thus, this is the first report of N. oryzae causing leaf spot of O. rufipogon in China. The finding provides the information important for further studies to develop management strategies for control of this disease.

First Report on Colletotrichum siamense Causing Anthracnose of Castor Bean in Zhanjiang, China.

Castor bean (Ricinus communis L.) is an important oil crop. Anthracnose lesions were observed on leaves of castor bean at the stage of budding and fruiting in field (21˚17'51''N, 110˚18'16''E), Zhanjiang, Guangdong Province, China in August 2019. The incidence rate was approximately 40% (n=600 investigated plants). Early symptoms were yellow spots appearing from the edge or the tip of the leaves. Later, the spots gradually expanded and became dark brown, which coalesced into larger irregular or circular lesions (Supplementary Figure 1). Seven diseased leaves were collected from seven plants. Margins of the diseased tissue were cut into 2 mm × 2 mm pieces. The surfaces were disinfested with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Thereafter, the samples were rinsed three times in sterile water, placed on PDA, and incubated at 28 °C. Pure cultures were obtained by transferring hyphal tips to new PDA plates. A single-spore isolate (RLC-1) was used for further study. The colony of isolate RLC-1 on PDA was white to gray in color with cottony mycelia in 6 days at 28 °C. Conidia were one-celled, hyaline, cylindrical, clavate, obtuse at both ends and measured 14.2 to 18.5 µm × 3.8 to 5.5 µm (n =50). Appressoria were oval to irregular in shape, dark brown, and ranged from 7.3 to 10.5 µm × 5.7 to 6.5 µm (n = 20). Morphological characteristics of isolate RLC-1 were consistent with the description of Colletotrichum siamense (Prihastuti et al. 2009; Sharma et al. 2013). DNA of the isolate RLC-1 was extracted for PCR sequencing using primers for the rDNA ITS (ITS1/ITS4), GAPDH (GDF1/GDR1), and ACT (ACT-512F/ACT-783R) (Weir et al. 2012). Analysis of the ITS (accession no. MN880199), GAPDH (MN884048), and ACT (MN891766) sequences revealed a 99%-100% identity with the corresponding ITS (JX010250), GAPDH (KX578786), ACT (JX009541) sequences of C. siamense in GenBank. A phylogenetic tree was generated on the basis of the concatenated data from sequences of ITS, GAPDH, and ACT that clustered the isolate RLC-1 with C. siamense with the type strain ICMP 19118 (Supplementary Figure 2). Morphological characteristic and phylogenetic analysis identified the isolate RLC-1 associated with anthracnose of castor bean as C. siamense. Pathogenicity test was performed in a greenhouse at 24 °C to 30 °C with 80% relative humidity. Twenty healthy plants of Zi Bi No. 5 castor bean (2 month old) were grown in pots with one plant in each pot. Inoculation was conducted on leaves with mycelial plugs of RLC-1 or agar plugs (as control). Three plugs were considered for each leaflet. Ten plants were used in each treatment (five for wounded inoculation and five for unwound inoculation). Anthracnose lesions as earlier were observed on the leaves after 2 weeks, while the control plants remained healthy. The pathogen re-isolated from all inoculated leaves was identical to the isolate RLC-1 by morphology and ITS analysis but not from control plants. C. siamense causes anthracnose on various plant hosts, including mango in Colombia (Pardo-De la Hoz et al. 2016) and Rosa chinensis in China (Feng et al. 2019) but not including castor bean. To the best of our knowledge, this study is the first to report C. siamense causing anthracnose on castor bean. Thus, this work provides a basis for focusing on the management of the disease in future.

First Report of Leaf Spot caused by Curvularia lunata on Wild Rice in China.

Wild rice (Oryza rufipogon), a species only recently cultivated in China, is an invaluable resource for rice breeding and basic research. In June 2019, a leaf spot disease on wild rice (O. rufipogon cv. 'Haihong-12') was observed in a 3.3 ha field in Zhanjiang (20.93 N, 109.79 E), China. The early symptoms were the presence of small, brown, and circular to oval spots that eventually turned reddish brown. The size of the spots varied from 1.0-5.0 mm × 1.0-3.0 mm. Disease incidence was higher than 20%. High temperature and high humidity climate were favorable for the disease occurrence. Twenty diseased leaves were collected from the field. The margin of the diseased tissues was cut into 2 mm × 2 mm pieces, surface-disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s, then rinsed three times with sterile water before isolation. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 °C in the dark for 4 days. Pure cultures were produced by transferring hyphal tips to new PDA plates. Three isolates, namely, Cls-1, Cls-2, and Cls-3, were subjected to further morphological and molecular studies. The colonies of the three isolates on PDA were initially light gray later becoming dark green. Conidiophores were erect, dark brown, geniculate, and unbranched. Conidia were fusiform, geniculate or hook-shaped, smooth-walled, dark-brown, 3-septate, with the second curved cell about 13.4-18.2 µm × 6.5-8.6 µm in size (n = 30). These morphological features agreed with previous descriptions of Curvularia lunata (Wakker) Boed (Macri and Lenna 1974). The ITS region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and translation elongation factor (EF-1α) were amplified using primers ITS1/ITS4, gpp1/gdp2 (Berbee et al. 1999), and EF-1/EF-2 (O'Donnell 1997), respectively. Amplicons of the three isolates were sequenced and submitted to GenBank (accession nos. MW042182, MW042183, and MW042184; MW091453, MW091454, and MW091455; MW090049, MW090050, and MW090051). The sequences of the two isolates were 100% identical to those of C. lunata (accession nos. MG971304, MG979801, MG979800) according to the results of BLAST analysis. A phylogenetic tree was built on the basis of concatenated data from the sequences of ITS, GAPDH, and EF-1α via the maximum likelihood method. The tree clustered Cls-1, Cls-2, and Cls-3 with C. lunata. The three isolates were determined as C. lunata by combining morphological and molecular characteristics. Pathogenicity tests were performed on Cls-1 in a greenhouse at 24 °C-30 °C with 80% relative humidity. Individual rice plants (cv. 'Haihong-12') with three leaves were grown in 10 pots, with approximately 50 plants per pot. Five pots were inoculated by spraying a spore suspension (105 spores/mL) onto leaves until runoff occurred, and another five pots were sprayed with sterile water and used as controls. The test was conducted three times. Disease symptoms were observed on the leaves after 10 days, but the controls remained healthy. C. lunata occurs on O. sativa (rice) (Liu et al. 2014; Majeed et al. 2016), but it has not been reported on O. rufipogon until now. To the best of our knowledge, this study is the first to report that C. lunata causes leaf spots on O. rufipogon in China. Thus, vigilance is required for breeding O. rufipogon.

First Report of Lasiodiplodia theobromae Causing Branch Dieback on Castor Bean in Zhanjiang, China.

Castor bean (Ricinus communis L.) is an oil crop of significant economic importance in the industry and medicine. In August 2019, a branch dieback disease was observed on castor bean in a field in Zhanjiang (21.17°N, 110.18°E), China. The incidence rate was 35% (n=600 investigated plants). Symptoms were discoloration of leaves, branch dieback, and discoloration of internal stem tissues. The disease had spread to the whole branches and causing the plant to die. Seven diseased branches were collected from seven plants. Margins between healthy and diseased tissues were cut into 2 mm × 2 mm pieces. The surfaces were disinfested with 75% ethanol for 30 s and 2% sodium hypochlorite for 60 s. Then, the samples were rinsed thrice in sterile water, placed on PDA, and incubated at 28 °C. Pure cultures were obtained by transferring the hyphal tips to new PDA plates. Eighteen isolates were obtained (the isolate rate of 75%), which were the same fungus on the basis of morphological characteristics and molecular analysis of the internal transcribed spacer (ITS). A single representative isolate (RiB-1) was used for further study. The colony of RiB-1 was 5 cm in diameter on the 5th day on the PDA culture. The colony was greenish gray with an irregularly distributed and fluffy aerial mycelium, which turned black after 10 days. The mature conidia were 21.3-26.5 µm × 12.2-15.7 µm in size (n=100) and had two ovoid, dark brown cells with longitudinal striations. The morphological characteristics of the colonies were consistent with the description of Lasiodiplodia sp. (Alves et al. 2008). Three regions of the ITS, translation elongation factor (EF1-α), and ß-tubulin genes were amplified and sequenced with the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Alves et al. 2008), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. The resulting sequences were deposited in the GenBank under accession numbers MN759432 (ITS), MN719125 (EF1-α), and MN719128 (ß-tubulin). BLASTn analysis demonstrated that these sequences were 100% identical to the corresponding ITS (MK530052), EF1-α (MK423878), and ß-tubulin (MN172230) sequences of L. theobromae. Based on the morphological and molecular data, RiB-1 was determined as L. theobromae. A pathogenicity test was performed in a greenhouse with 80% relative humidity at 25 °C to 30 °C. Ten healthy plants of Zi Bi No. 5 castor bean (1-month-old) were grown in pots with one plant in each pot. Five pots were wound-inoculated with 5-mm-diameter mycelial plugs obtained from 7-day cultures. Five additional pots treated with PDA plugs served as the controls. Inoculated stems were moisturized with sterile cotton for five days. The test was conducted three times. Disease symptoms, similar to those in the field, were observed on the inoculated plants two weeks after inoculation, and L. theobromae was 100% reisolated from the inoculated plants. The control plants remained symptomless, and reisolations were unsuccessful. These results consistent with Koch's postulates. L. theobromae (Lima et al. 1997) and L. hormozganensis (Fábio et al. 2018) had been reported to cause stem rot on castor bean in Brazil, but whether L. theobromae caused the branch dieback on castor bean in China has not been reported yet. Thus, this study is the first report of L. theobromae causing the branch dieback on castor bean in Zhanjiang, China. This study provides an important reference for the control of the disease.

First Report of Colletotrichum siamense Causing Anthracnose of Monstera deliciosa in Zhanjiang, China.

Monstera deliciosa Liebm is an ornamental foliage plant (Zhen et al. 2020De Lojo and De Benedetto 2014). In July of 2019, anthracnose lesions were observed on leaves of M. deliciosa cv. Duokong with 20% disease incidence of 100 plants at Guangdong Ocean University campus (21.17N,110.18E), Guangdong Province, China. Initially affected leaves showed chlorotic spots, which coalesced into larger irregular or circular lesions. The centers of spots were gray with a brown border surrounded by a yellow halo (Supplementary figure 1). Twenty diseased leaves were collected for pathogen isolation. Margins of diseased tissue was cut into 2 × 2 mm pieces, surface-disinfected with 75% ethanol for 30 s and 2% sodium hypochlorite (NaOCl) for 60 s, rinsed three times with sterile water before isolation. Potato dextrose agar (PDA) was used to culture pathogens at 28℃ in dark. Successively, pure cultures were obtained by transferring hyphal tips to new PDA plates. Fourteen isolates were obtained from 20 leaves. Three single-spore isolates (PSC-1, PSC-2, and PSC-3) were obtained ,obtained, which were identical in morphology and molecular analysis (ITS). Therefore, the representative isolate PSC-1 was used for further study. The culture of isolate PSC-1 on PDA was initially white and later became cottony, light gray in 4 days, at 28 °C. Conidia were single celled, hyaline, cylindrical, clavate, and measured 13.2 to 18.3 µm × 3.3 to 6.5 µm (n = 30). Appressoria were elliptical or subglobose, dark brown, and ranged from 6.3 to 9.5 µm × 5.7 to 6.5 µm (n = 30). Morphological characteristics of isolate PSC-1 were consistent with the description of Colletotrichum siamense (Prihastuti et al. 2009; Sharma et al. 2013). DNA of the isolate PSC-1 was extracted for PCR sequencing using primers for the rDNA ITS (ITS1/ITS4), GAPDH (GDF1/GDR1), ACT (ACT-512F/ACT-783R), CAL (CL1C/CL2C), and TUB2 (ßT2a/ßT2b) (Weir et al. 2012). Analysis of the ITS (accession no. MN243535), GAPDH (MN243538), ACT (MN512640), CAL (MT163731), and TUB2 (MN512643) sequences revealed a 97-100% identity with the corresponding ITS (JX010161), GAPDH (JX010002), ACT (FJ907423), CAL (JX009714) and TUB2 (KP703502) sequences of C. siamense in GenBank. A phylogenetic tree was generated based on the concatenated sequences of ITS, GAPDH, ACT, CAL, and TUB2 which clustered the isolate PSC-1 with C. siamense the type strain ICMP 18578 (Supplementary figure 2). Based on morphological characteristics and phylogenetic analysis, the isolate PSC-1 associated with anthracnose of M. deliciosa was identified as C. siamense. Pathogenicity test was performed in a greenhouse at 24 to 30oC with 80% relative humidity. Ten healthy plants of cv. Duokong (3-month-old) were grown in pots with one plant in each pot. Five plants were inoculated by spraying a spore suspension (105 spores ml-1) of the isolate PSC-1 onto leaves until runoff, and five plants were sprayed with sterile water as controls. The test was conducted three times. Anthracnose lesions as earlier were observed on the leaves after two weeks, whereas control plants remained symptomless. The pathogen re-isolated from all inoculated leaves was identical to the isolate PSC-1 by morphology and ITS analysis, but not from control plants. C. gloeosporioides has been reported to cause anthracnose of M. deliciosa (Katakam, et al. 2017). To the best of our knowledge, this is the first report of C. siamense causing anthracnose on M. deliciosa in ChinaC. siamense causes anthracnose on a variety of plant hosts, but not including M. deliciosa (Yanan, et al. 2019). To the best of our knowledge, this is the first report of C. siamense causing anthracnose on M. deliciosa, which provides a basis for focusing on the management of the disease in future.

Effects of gastric cancer cells on the differentiation of Treg cells.

The aim of this study was evaluated the prevalence of Treg cells in peripheral blood in patients with gastric cancer, and investigate the effect of gastric cancer cells on their differentiation. ELISA was employed to assess the concentrations of TGF-ß and IL-10 in gastric cancer patients' serum. Then, mouse gastric cancer cells were co-cultured with T lymphocytes or T lymphocytes + anti-TGF-ß. Flow cytometric analysis and RT-PCR were then performed to detect Treg cells and TGF-ß and IL-10 expression in gastric cancer cells. Our data showed that the expression of TGF-ß and IL-10 in the patients with gastric cancer was increased compared to the case with healthy donors. The population of Treg cells and the expression levels of TGF-ß and IL-10 in the co-culture group were much higher than in the control group (18.6% vs 9.5%) (P<0.05). Moreover, the population of Treg cells and the expression levels of TGF-ß and IL-10 in the co-culture systerm were clearly decreased after addition of anti-TGF-ß (7.7% vs 19.6%) (P<0.01). In conclusion, gastric cancer cells may induce Treg cell differentiation through TGF-ß, and further promote immunosuppression.

[Analysis of full-length gene sequence of rabies vaccine virus aG strain].

To sequence and analyze the full-length gene sequence of rabies vaccine virus aG strain. The full-length gene sequence of aG strain was amplified by RT-PCR by 8 fragments,each PCR product was cloned into vector pGEM-T respectively, sequenced and assemblied; The 5' leader sequence was sequenced with method of 5' RACE. The homology between aG and other rabies vaccine virus was analyzed by using DNAstar and Mega4. 0 software. aG strain was 11 925nt(GenBank accession number: JN234411) in length and belonged to the genotype I . The Bioinformatics revealed that the homology showed disparation form different rabies vaccine virus. the full-length gene sequence of rabies vaccine virus aG strain provided a support for perfecting the standard for quality control of virus strains for production of rabies vaccine for human use in China.

[Analysis of full-length gene sequence of a rabies vaccine strain CTN-1 for human use in China].

CTN-1 is one of the rabies vaccine strains for human use in China, but there has been no report on the full-length gene sequence of CTN-1. In this study, the full-length gene of CTN-1 was amplified by RT-PCR, each PCR product was cloned into T vector and then sequenced, assemblied and compared with other vaccine strains as well as the wild Chinese rabies isolates. The phylogenetic tree of G gene was constructed and the genetic homology was analyzed. The results revealed that CTN-1 was 11 925nt (GenBank accession number: FJ959397)in length and belonged to the genotype I. The full-length nucleotide homologies among CTN-1 and other rabies virus strains were between 81.5%-93.4%, of which the lowest 81.5% was between CTN-1 strain and bat isolate SHBRV, and the highest 93.4% was between CTN-1 and Chinese isolate HN10. The phylogenetic analysis revealed that the majority of Chinese isolates could be grouped into the same clade with the CTN-1 strain, but aG and some vaccine strains from abroad such as Flury, PM, PV, ERA, RC-HL and a few Chinese strains were grouped in another clade. Comparsion of the G protein genes also showed that the homologies among CTN-1 and most of the Chinese isolates were higher than that of the other vaccine strains to those Chinese strains. Therefore, it suggests that the CTN-1 strain is more suitable and rational to be used for the production of rabies inactivated vaccine in China than the others.

[Study on the feasibility of a cell-culture inoculation test in the detection and isolation of street rabies virus].

OBJECTIVE: Feasibility of using MNA cell-culture inoculation test to detect and isolate the street rabies virus. METHODS: Using MNA cell-culture inoculation test, fluorescent antibody test (FAT) and sandwich ELISA with double-antibodies to detect 33 specimens of street rabies virus, 20 specimens of negative canine brains and 4 specimens of healthy mice brains. RESULTS: 33 specimens of street rabies virus were positive to the cell-culture inoculation test but the others were negative. The concordances of MNA cell-cultured inoculation test with FAT and sandwich ELISA with double-antibodies were both 100%. CONCLUSION: MNA cell-culture inoculation test appeared to be both highly sensitive and specific in detecting the street rabies virus, and could be used in detection and isolation of the virus.