Rice-Magnaporthe oryzae interactions in resistant and susceptible rice cultivars under panicle blast infection based on defense-related enzyme activities and metabolomics.

Rice blast, caused by rice blast fungus (Magnaporthe oryzae), is a global threat to food security, with up to 50% yield losses. Panicle blast is a severe form of rice blast, and disease responses vary between cultivars with different genotypes. Reactive oxygen species (ROS)-mediated signaling reactions and the phenylpropanoid pathway are important defense mechanisms involved in recognizing and resisting against fungal infection. To understand rice-M. oryzae interactions in resistant and susceptible cultivars, we determined dynamic changes in the activities of five defense-related enzymes in resistant cultivar jingsui 18 and susceptible cultivar jinyuan 899 infected with M. oryzae from 4 to 25 days after infection. We then performed untargeted metabolomics analyses to profile the metabolomes of the cultivars under infected and non-infected conditions. Dynamic changes in the activities of five defense-related enzymes were closely related to panicle blast resistance in rice. Metabolome data analysis identified 634 differentially accumulated metabolites (DAMs) between resistant and susceptible cultivars following infection, potentially explaining differences in disease response between varieties. The most enriched DAMs were associated with lipids and lipid-like molecules, phenylpropanoids and polyketides, organoheterocyclic compounds, organic acids and derivatives, and lignans, neolignans, and related compounds. Multiple metabolic pathways are involved in resistance to panicle blast in rice, including biosynthesis of other secondary metabolites, amino acid metabolism, lipid metabolism, phenylpropanoid biosynthesis, arachidonic acid metabolism, arginine biosynthesis, tyrosine metabolism, tryptophan metabolism, tyrosine and tryptophan biosynthesis, lysine biosynthesis, and oxidative phosphorylation.

Comparison of Transcriptome between Tolerant and Susceptible Rice Cultivar Reveals Positive and Negative Regulators of Response to Rhizoctonia solani in Rice.

Rice (Oryza sativa L.) is one of the world's most crucial food crops, as it currently supports more than half of the world's population. However, the presence of sheath blight (SB) caused by Rhizoctonia solani has become a significant issue for rice agriculture. This disease is responsible for causing severe yield losses each year and is a threat to global food security. The breeding of SB-resistant rice varieties requires a thorough understanding of the molecular mechanisms involved and the exploration of immune genes in rice. To this end, we conducted a screening of rice cultivars for resistance to SB and compared the transcriptome based on RNA-seq between the most tolerant and susceptible cultivars. Our study revealed significant transcriptomic differences between the tolerant cultivar ZhengDao 22 (ZD) and the most susceptible cultivar XinZhi No.1 (XZ) in response to R. solani invasion. Specifically, the tolerant cultivar showed 7066 differentially expressed genes (DEGs), while the susceptible cultivar showed only 60 DEGs. In further analysis, we observed clear differences in gene category between up- and down-regulated expression of genes (uDEGs and dDEGs) based on Gene Ontology (GO) classes in response to infection in the tolerant cultivar ZD, and then identified uDEGs related to cell surface pattern recognition receptors, the Ca2+ ion signaling pathway, and the Mitogen-Activated Protein Kinase (MAPK) cascade that play a positive role against R. solani. In addition, DEGs of the jasmonic acid and ethylene signaling pathways were mainly positively regulated, whereas DEGs of the auxin signaling pathway were mainly negatively regulated. Transcription factors were involved in the immune response as either positive or negative regulators of the response to this pathogen. Furthermore, our results showed that chloroplasts play a crucial role and that reduced photosynthetic capacity is a critical feature of this response. The results of this research have important implications for better characterization of the molecular mechanism of SB resistance and for the development of resistant cultivars through molecular breeding methods.

First Report of Colletotrichum aenigma Causing Anthracnose on Bromus japonicus in Tianjin, China.

Japanese brome (Bromus japonicus Thunb.) is a weed commonly found in roadsides, floodplain wetlands, and farmlands. During September 2020 and 2021, a leaf spot disease was observed on B. japonicus in greenhouses of Baodi district, Tianjin, China (117°15'E, 39°47'N). More than 10% of the weeds were infected. Initial irregular brown spots on leaf apices continued to expand until adjacent spots coalesced. Eventually, severely infected leaves became yellow, thinner, drier and withered. Small patches (3×3 mm) were cut from symptomatic leaves, sterilized with 75% ethanol for 30s, rinsed three times with sterile water and incubated on Petri dishes with potato dextrose agar at 25°C in darkness for 7 days. Three isolates, with uniform morphology were selected for further analysis. Colonies were cottony with entire edges and aerial white mycelia; and average growth rate of 4.5 mm/day. The upper side was pale white, and the reverse side was grayish-green. Conidia were aseptate, hyaline, subcylindrical with rounded ends, 8.6 to 18.7×4.4 to 8.3 µm (n = 50). Appressoria were dark brown, oval or irregular shaped with a few lobes, 5.7 to 9.4×4.5 to 7.8 µm (n = 50). Total genomic DNA of isolates was extracted with Fungal DNA Kit (GBCBIO, Guangzhou, China). Primers for sequences of internal transcribed spacer (ITS) regions, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß-tubulin (TUB2), and calmodulin (CAL) genes were amplified and sequenced (Weir et al., 2012). After aligned and trimmed, the sequences of TJBDA1, TJBDA2, and TJBDA3 were identical. TJBDA1 representative isolate sequences were deposited in GenBank ITS OP247554 with 99.83% (576/577) similarity to MT476809, GAPDH OP414834 with 99.59% (244/245) similarity to MT501009, TUB2 OP414836 with 100% (703/703) to MT501053, and CAL OP414835 with 100% (601/601) to MT500921. Maximum likelihood trees based on concatenated sequences of the four genes were constructed using MEGA7.0. The results showed that the strains isolated from B. japonicus were closely related to C. aenigma with 99% bootstrap support. Pathogenicity tests were conducted on 3-leaf stage B. japonicus seedlings. Conidial suspension of TJBDA1 (1×106 conidia/ml) brushed from a 7-day-old culture of the fungus were sprayed on 9 B. japonicus seedlings. Control plants were sprayed with sterile water. All treatments were replicated four times. The treatment plants were placed in an incubator (25°C, relative humidity > 80%, 12-h photoperiod). Typical leaf spot symptoms resembling ones in the fields were observed on inoculated leaves after 7 days, but control leaves remained symptomless. The fungi reisolated from diseased leaves were morphologically and molecularly identical to the inoculated isolatescompleting Koch's postulates. According to morphological, pathological characteristics and multilocus phylogenetic analysis, the isolated strains from B. japonicus were identified as C. aenigma. To our knowledge, this is a new host record for C. aenigma causing anthracnose on B. japonicus in China. Currently, B. japonicus has evolved a high level of resistance to herbicides in some regions of China (Li et al, 2022) and C. aenigma caused serious disease to B. japonicus. We hope to discover a biocontrol method against weed on non-host cultivated plants through the production of secondary metabolites by C. aenigma.

Complete Genome Sequence Resource for Cercospora apii Causing Leaf Spot of Celery.

Cercospora apii is an important seedborne pathogenic fungus causing severe Cercospora leaf spot of celery worldwide. Here, we first present a complete genome assembly of C. apii QCYBC from celery, based on Illumina paired-end and PacBio long-read sequencing data. The high-quality genome assembly contains 34 scaffolds with a 34.81 Mb genome size, 330 interspersed repeat genes, 114 noncoding RNAs, and 12,631 protein-coding genes. The benchmarking universal single-copy ortholog (BUSCO) analysis indicated that 98.2% of the BUSCOs were complete, whereas 0.3, 0.7, and 1.1% were duplicated, fragmented, and missing, respectively. Based on annotation, 508 carbohydrate-active enzymes, 243 cytochromes P450 enzymes, 1,639 translocators, 1,358 transmembrane proteins, and 1,146 virulence genes were identified. This genome sequence provides a valuable reference for future studies to improve understanding of the C. apii-celery pathosystem. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Identification of Strawberry Wilt Caused by Plectosphaerella cucumerina in China.

Strawberry (Fragaria × ananassa Duch.) is a widely cultivated economic crop in China. In April 2022, an unusual wilt disease was observed on strawberry plants (6 months' old) in Chenzui town, Wuqing district, Tianjin, China (117°1'E, 39°17'N). The incidence across the greenhouses (≈0.34 ha) was approximately 50 to 75%. The first wilt symptoms were observed on the outer leaves, then the whole seedlings wilted and died. The rhizome of the diseased seedlings changed color and became necrotic and rotted. Symptomatic roots were surface disinfected with 75% ethanol for 30 s, washed with sterile distilled water for three times, and then cut into 3 mm2 pieces (four pieces per seedling) and placed on petri dish with potato dextrose agar (PDA) containing 50 mg/L of streptomycin sulfate and incubated at 26℃ in the dark. After 6 days' incubation, hyphal tips of the growing colonies were transferred onto PDA. Eighty-four isolates belong to five fungal species were obtained from 20 diseased root samples based on their morphological characteristics. To confirm their pathogenicity, ten two-month-old healthy seedlings of strawberry (cv Red Face) planted in sterilized nutrient soil, were inoculated by pouring 50 mL of conidial suspension (107 conidia/mL) (Cai et al. 2021). Another ten seedlings poured with sterile distilled water were used as controls. Each treatment was repeated three times in a greenhouse at 25 to 28℃ and 75% relative humidity under a 12-h photoperiod. After 15 days，only seedlings inoculated with Plectosphaerella (an original percentage of 35.71%) exhibited similar symptoms to those of diseased seedlings originally observed in the field. Seedlings had no symptoms in the control and other fungi inoculation treatments. To fulfill the Koch's postulates, Plectosphaerella isolates were reisolated from each inoculated, symptomatic seedling with a percentage of 100%, but were not recovered from any of the control seedlings. The experiments were repeated twice with similar results. The results indicated that the genus Plectosphaerella was the pathogen causing strawberry wilt. Colonies of the genus Plectosphaerella isolates on PDA were white to cream, and then gradually became salmon pink, with few aerial hyphae and slimy surfaces. Colonies produced numerous hyphal coils with conidiophores. Conidia were 4.56 to 10.07 µm × 1.11 to 4.54 µm (avg. 7.10 × 2.56 µm, n=100), septate or aseptate, ellipsoidal, hyaline and smooth. Such morphological characteristics were identical to those of Plectosphaerella spp. (Palm et al. 1995). For species identification, the ITS region and D1/D2 domain of the 28S rRNA gene of representative isolates (CM2, CM3, CM4, CM5 and CM6) were amplified and sequenced with the primer pair ITS1/ITS4 and NL1/NL4, respectively (White et al. 1990; O'Donnell and Gray 1993). By BLASTn analysis, the obtained sequences of ITS amplicon (ON629742, ON629743, ON629744, ON629745, and ON629746) and D1/D2 domain amplicon (OQ519896, OQ519897, OQ519898, OQ519899, and OQ519900) showed 99.14% to 99.81% identity to sequences of P. cucumerina (MW320463.1 and HQ239025.1) in the NCBI database. A multilocus phylogenetic tree performed by the UPGMA analysis showed that the representative isolates were assigned to the group of P. cucumerina. To our knowledge, this is the first report of P. cucumerina causing strawberry wilt worldwide. This disease may induce serious economic losses in strawberry production, thus effective management strategies should be taken.

Effect of High-Frequency rTMS combined with bilateral arm training on brain functional network in patients with chronic stroke: An fNIRS study.

OBJECTIVE: Neurological evidence for the combinational intervention coupling rTMS with motor training for stroke rehabilitation remains limited. This study aimed to investigate the effects of rTMS combined with bilateral arm training (BAT) on the brain functional reorganization in patients with chronic stroke via functional near-infrared spectroscopy (fNIRS). METHODS: Fifteen stroke patients and fifteen age-matched healthy participants were enrolled and underwent single BAT session (s-BAT) and BAT immediately after 5-Hz rTMS over the ipsilesional M1 (rTMS-BAT), measured cerebral haemodynamics by fNIRS. Functional connectivity (FC), the clustering coefficient (Ccoef), and local efficiency (Eloc) were applied to evaluate the functional response to the training paradigms. RESULTS: The differences in FC responses to the two training paradigms were more pronounced in stroke patients than in healthy controls. In the resting state, stroke patients exhibited significantly lower FC than controls in both hemispheres. rTMS-BAT induced no significant difference in FC between groups. Compared to the resting state, rTMS-BAT induced significant decreases in Ccoef and Eloc of the contralesional M1 and significant increases in Eloc of the ipsilesional M1 in stroke patients. Additionally, these above two network metrics of the ipsilesional motor area were significantly positively correlated with the motor function of stroke patients. CONCLUSIONS: These results suggest that the rTMS-BAT paradigm had additional effects on task-dependent brain functional reorganization. The engagement of the ipsilesional motor area in the functional network was associated with the motor impairment severity of stroke patients. fNIRS-based assessments may provide information about the neural mechanisms underlying combination interventions for stroke rehabilitation.

First Report of Stem and Root Rot Caused by Fusarium oxysporum on Geranium in China.

Geranium (Pelargonium hortorum) is a widely cultivated ornamental plant in the world. In August 2021, geranium plants exhibited stem and root rot symptoms in a garden in Tianjin, China (117°20'E, 39°13'N) with an approximate incidence of 10%. Symptoms were rotted stem and root, chlorotic leaves, wilting, discolored vascular tissues and eventually plant death. White mycelia were observed growing on the surface of the infected stems and roots in a high humidity environment. Symptomatic plants were collected from three nurseries (20 plants per nursery) in the Tianjin garden, out of which 10 plants were arbitrarily selected for pathogen isolation. The diseased stem and root were rinsed under tap water, blotted dry, and excised into 3 mm2 pieces (three pieces per plant) at the boundary of the symptomatic area. The pieces were surface disinfected with 75% ethanol for 30 s, washed with sterile distilled water for three times, and then placed on potato dextrose agar (PDA) containing 50 mg/L of streptomycin sulfate and incubated at 26℃ in the dark. After 6 days, 24 of 30 isolates generated white to pale pink aerial hyphae on PDA. Macroconidia were slightly curved at apex, hyaline and falcate, 3 to 5 septate, 19.2 to 41.08 µm in length and 2.95 to 5.62 µm in width (n = 30). Microconidia were single-celled, hyaline, non-septate and ovoid, and measured 4.98 to 16.48 µm in length and 1.58 to 4.69 µm in width (n = 30). These characteristics matched the description of Fusarium oxysporum (Leslie and Summerell 2006). The ITS gene, TEF1-α gene, mtSSU gene and RPB2 gene of representative isolates (T7, T8 and T9) were amplified and sequenced for species identification (Carbone and Kohn 1999; Li et al. 1994; Miller and Huhndorf 2005). BLASTn analyses of the ITS amplicon (OK326801, OK326789 and OK326823), TEF1-α amplicon (OM974708, OM974710 and OM974711), mtSSU amplicon (OK330481, OM948931 and OM948932) and RPB2 amplicon (OM974709, OM974712 and OP331332) obtained with cognate sequences available in GenBank showed 99.31% to 100% identity to sequences of F. oxysporum in the NCBI database and the Fusarium MLST database. The multilocus phylogenetic analysis from the Fusarium MLST database performed by UPGMA showed that the representative isolates were assigned to the group of F. oxysporum. Ten two-month-old healthy plants of geranium (cv. Horizon Scarle) were inoculated by dipping the roots into a conidial suspension of 1×107 conidia/mL for 10 min (Monther et al. 2021). Another ten plants dipped in sterile distilled water served as controls. After inoculated or noinoculated treatments, plants were planted in sterilized nutrient soil in plastic pots (one plant per pot). Each treatment was repeated three times in a greenhouse at 28℃ and 75% relative humidity. Two weeks later, inoculated plants exhibited similar symptoms to those of diseased plants in the garden. Whereas the control plants remained healthy. To fulfill the Koch's postulates, F. oxysporum was reisolated from each inoculated, symptomatic plant, but was not recovered from any of the control plants. The experiments were repeated twice with similar results. F. oxysporum is reported to cause disease on Pelargonium graveolens in Kenya (Nattrass 1961) and on Pelargonium sp. in California (French 1989), to our knowledge, this is the first report of F. oxysporum causing stem and root rot on Pelargonium hortorum in China. Identification of disease caused by F. oxysporum on geranium will guide disease management.

First Report of Colletotrichum truncatum Causing Anthracnose of Green Foxtail (Setaria viridis) in China.

Green foxtail (Setaria viridis (L.) Beauv.), belonging to the family Gramineae, is a monocotyledonous plant that is distributed in tropical and subtropical regions of the world. S. viridis is one of the most abundant weeds in corn, soybean, rice and other major crops in China, which competes with crops for light, moisture and nutrients, leading to yield losses. In September 2021, an unknown leaf spot disease was observed on the leaves of S. viridis in many greenhouses of Xinkou town, Xiqing district, Tianjin, China (116.95729, 39.09088), under cloudy and high humid conditions after a week of rain. Over 60% of the weeds were observed with leaf spots in 28 greenhouses of XinKou town. The characteristics of the disease were observed and investigated. Initial symptoms were brown spots of 1 to 5 mms, longitudinal elliptic, round, or spindle-shaped lesions on leaves of S. viridis. These spots continued to spread shortly after the onset of the symptoms. At the late-stage disease, the spots' edges were dark brown and irregular. Eventually, the center of the spots turned grayish-white and became thinner and drier until fracture. To investigate the disease, symptomatic weed leaves were separated and small patches with infected spots were cut out. Diseased tissues (3×3 mm) were disinfected with 75% alcohol for 30s  35s, rinsed three times with sterile distilled water and then placed on potato dextrose agar (PDA) at 25°C with a 12-h photoperiod for 7 days in incubators (RXZ-280C, Ningbo, China). With the pathogen growing on the PDA, three mycelia with uniform morphology were observed, which were named SVCT-01, SVCT-02, and SVCT-03, respectively. These mycelia were transferred and cultured for daily observation. The color of these mycelia on PDA appeared gray at first, which eventually turned to grayish black with numerous black microsclerotia, setae, and a few aerial mycelia after 7 days. The setae were 75 to 120 ×3.5 to 5 µm, with elliptic to claviform appressoria. Conidia were hyaline, falcate, unicellular, 16 to 25 × 2.6 to 3.8 µm (n=50). All characteristics of isolates were consistent with the description of Colletotrichum truncatum (Sutton, 1992). Pathogenicity testing was conducted on 3-leaves-stage S. viridis seedlings. Conidial suspension (106 conidia/mL) of isolates were sprayed on 20 S. viridis seedlings with the suspension of each isolate was sprayed on 10 seedlings. Ten seedlings sprayed with sterilized distilled water were used as the control. Three replicates were performed on each treatment. The treatment plants were maintained in the incubator (25°C, relative humidity > 80%, 12-h photoperiod). Typical leaf spot symptoms were observed on inoculated leaves after 7 days, the control leaves remained symptomless. The fungus reisolated from the lesions of diseased leaves were morphological and molecularly identical to the inoculated isolates. The results echo with Koch's postulates，suggesting that the obtained isolates SVCT-01, SVCT-02 and SVCT-03 are potential pathogen in Setaria viridis. To confirm the species' identity, total genomic DNA of isolates were extracted using a Fungal DNA Kit (GBCBIO, Guangzhou, China). Sequences of internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), and ß-tubulin (TUB2) regions were identified via PCR (Guerber et al, 2003; Weir et al, 2012). The sequences of SVCT-01, SVCT-02 and SVCT-03 showed more than 99% homology with Colletotrichum truncatum strains CBS:151.35 (GenBank Accession No. GU227862, GU228254, GU227960, GU228156) (Damm, 2009). The sequences of SVCT-01 were deposited in GenBank as a representative isolate under the accession numbers OL629177, OL627527, OM040388, OM040389. Maximum likelihood trees based on concatenated sequences of the four genes were constructed using MEGA7.0. The results showed that the strains isolated from Setaria viridis were closely related to Colletotrichum truncatum with 100% bootstrap support. According to morphological, pathological characteristics, and multilocus phylogenetic analysis, the isolated strains (SVCT-01, SVCT-02 and SVCT-03) from S. viridis were identified as Colletotrichum truncatum (Weir et al, 2012). Colletotrichum sp. is a significant plant pathogen that was previously reported causing anthracnose on Setaria sp. Up to now, it has been reported that C. graminicola has infected nine species of Setaria sp. Such as Setaria glauca in New Zealand (Pennycook, 1989) and Setaria pumila in Zimbabwe (Lenne, 1990). In 1979s, C.graminicola was obtained from Setaria lutescens in China (Tai, 1979). To our knowledge, this is a new host record for Colletotrichum truncatum causing anthracnose on S. viridis in China. Colletotrichum truncatum spread rapidly and caused serious disease to Setaria viridis. We hope to discovery a biocontrol method against weed on non-host cultivated plants through the production of secondary metabolites by C. truncatum.

First report of Leaf Spot Caused by Paramyrothecium foliicola on Tomato (Solanum lycopersicum) in China.

Tomato (Solanum lycopersicum) is a staple vegetable across the world. In October 2019, leaf spots were observed on tomato (cv. Tianmi) in a greenhouse in JiZhou District Tianjin, China(117°10 'E; 39°55 'N). Symptoms initially appeared as small brown spots, which gradually expanded and turned into circular, oval or irregular spots (some spots with distinct concentric zones). In severe cases, some spots coalesced and eventually covered the whole leaf. Disease incidence ranged between 12 and 18%. Twenty symptomatic leaves from five plants were collected and cut into small pieces, surface disinfested in 2% NaClO for 60 s, rinsed three times in sterile water, and subsequently plated on potato dextrose agar (PDA). Plates were incubated at 25°C in the dark for 7 days. A total of 102 isolates were obtained and 92 isolates had the same morphology. Colonies were initially white with abundant aerial mycelia and formed sporodochia with conidial masses in olivaceous green concentric rings. All isolates formed single-celled, hyaline, and rod-shaped conidia were 4.91 to 7.43 (avg. 6.53±0.72) × 1.41 to 2.45 （avg. 2.11±0.30）µm with rounded ends (n=50). Conidiophores were highly branched. These characteristics resembled a Paramyrothecium-like fungus (Lombard et al. 2016). The genomic DNA of three representative single-spored isolates TJJXPF1-3 were extracted and the internal transcribed spacer (ITS) region, ß-tubulin (tub2), large subunit ribosomal RNA (LSU), calmodulin (cmdA) and translation elongation factor 1-alpha (tef1) genes were amplified and sequenced using the primer pairs ITS4/ITS5 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), LR0R/LR5 (Rehner and Samuels 1995; Vilgalys and Hester 1990), CAL-228F/CAL2Rd (Carbone and Kohn 1999; Groenewald et al. 2013) and EF1-728F/EF2 (O'Donnell et al. 1998), respectively. All sequences were deposited in GenBank (ITS: MW463444, OM368178, OM368179; tub2: MW269542，OM714930，OM714931; LSU: OM349050, OM397398, OM390582; cmdA: MW280443, OM350474, OM350476; tef1: MW560083, OM350475, OM350477). BLASTN analysis showed 99.3-100% similarity with reference isolate QB1 of P. foliicola (MK335967, MT415353, MT415362, MT415356 and MT415359). Multilocus phylogenetic analysis showed that TJJXPF1-3 best grouped with the P. foliicola clade, which was identified by morphological characteristics and phylogenetic analysis. To fulfill Koch's postulates, pathogenicity tests were conducted by spray-inoculation with a conidial suspension of isolate TJJXPF1 prepared with distilled water (1×105 conidia/mL) on five 45-day old tomato plants. Three healthy plants were sprayed with sterile water as control. All treatments were incubated in an artificial climate chamber (25°C, 80% RH, 12h light/12h dark ). After two weeks, leaf spots were observed on all inoculated plants, which were similar to those in the greenhouse of JiZhou District, while control plants remained asymptomatic. Additionally, the pathogens were reisolated from symptomatic leaves and three representative isolates TJJXPF4-6 were identified as P. foliicola. The pathogenicity tests were repeated thrice. To our knowledge, this is the first report of leaf spot caused by P. foliicola on tomato in China. This disease could be a serious threat to tomato production in the future. Our findings will help to differentiate this disease from other leaf spot-like diseases and develop disease control strategies.

Stemphylium lycopersici Causing Leaf Spot of Watermelon (Citrullus lanatus) in China.

Watermelon (Citrullus lanatus) is an important cucurbit crop in China. During September 2020, an unknown leaf spot disease was observed on watermelon in two greenhouses (640m2 per greenhouse) of Sangzi town, Jizhou district, in Tianjin, China (117°10'E, 39°55'N), where approximately 10% of plants were infected. Disease symptoms began as small, circular, brown spots on leaves. As these spots increased in size, they developed confluent, irregular lesions surrounded by dark brown edges. Severely affected plants had many wilted leaves followed by defoliation. Ten symptomatic leaves were collected for pathogen isolation. Diseased tissues (3×3 mm) were cut from the margins of lesions and surface disinfected with 1% NaClO for 1 min, rinsed three times with sterile distilled water and then placed on potato dextrose agar (PDA) at 25±2°C with a 12-h photoperiod for 7 to 10 days. Seven morphologically similar isolates were obtained from the ten infected leaves and purified by single-spore culturing for further study. The initial growth of the isolates on PDA appeared grayish white in obverse and bright yellow pigmentation in reverse. Colony color gradually deepened to grayish brown in obverse and brownish red in reverse. Conidia (n=50) were solitary, light brown, oblong to long elliptic, pointed or obtusely rounded at the top, constricted at the transverse septum, with verrucous processes on the surface, 36.3 to 64.2×16.6 to 25.1 µm, and the L/W ratio of conidia was 1.5-2.5. All characteristics were consistent with the description of Stemphylium lycopersici (Ellis 1971; Woudenberg et al. 2017). Total genomic DNA was extracted from a representative isolate (XG2-2) using a Fungal DNA Kit (GBCBIO, Guangzhou, China). The internal transcribed spacer (ITS) and translation elongation factor 1-α (EF1-α) genes (Sun et al. 2015) were amplified and sequenced with the primer pairs ITS1/ITS4 (5'-TCCGTAGGTGAACCTGCGG-3'/5'-TCCTCCGCTTATTGATATGC-3') and EF-1α-F/EF-1α-R(5'-TCACTTGATCTACAAGTGCGGTGG-3'/5'-CGATCTTGTAGACATCCTGGAGG-3'), respectively. The two sequences of strain XG2-2 (GenBank Accession No. MW362344 and MW664941) showed 100% and 99% identity to S. lycopersici strain 01 and strain KuNBY1 (GenBank Accession No. KR911814 and AB828256) respectively. The phylogenetic analysis using MEGA7 based on the sequences of ITS and EF1-α regions showed that the isolate XG2-2 was clustered with S. lycopersici isolates (strain 01 and strain KuNBY1). For the pathogenicity test, a spore suspension (1×106 spores/ml) in sterile distilled water from a 7-day-old culture of the fungus grown on PDA and counted with a hemacytometer was sprayed on leaves and stems of five healthy watermelon plants, grown for 2 months in the greenhouse at 25 to 30 °C, with 85% relative humidity. Conditions remained the same for inoculation experiments. Negative controls were healthy plants inoculated with sterile distilled water. The experiment was repeated twice. Six days after inoculation, typical leaf spot symptoms were observed on inoculated leaves, whereas control leaves remained symptomless. To satisfy Koch's postulates, the causal fungus was re-isolated from the lesions of inoculated plants, with morphological and cultural characteristics identical with the original isolate. Stemphylium lycopersici is a common fungus with a relatively extensive host range (Kee et al. 2018). In recent years, new host plants infected by S. lycopersici have been reported in Asia including Physali (Yange et al. 2020), common bean (Li et al. 2019), and potato (Kee et al. 2018). To our knowledge, this is a new host record for S. lycopersici causing leaf spot on watermelon in China. Sangzi watermelon is a special local product in the Jizhou district of Tianjin. At present the cultivated area in 1000 ha including 667 ha in controlled conditions and 333 ha of field-grown plants with a total annual output of 45000 Mg. In this survey, we found the disease caused by S. lycopersici on watermelon only in these two greenhouses, but cannot rule out the possibility of large-scale spread in the future. Therefore, integrated management strategies for this fungus need to be developed to reduce economic losses in commercial cultivation.

Genome Sequence Resource for Nigrospora oryzae, an Important Pathogenic Fungus Threatening Crop Production.

Nigrospora oryzae is an important phytopathogenic fungus with a broad host range. Here, we report an annotated draft of the genome of N. oryzae field strain GZL1 collected from maize assembled from PacBio and Illumina sequencing reads. The assembly we obtained has 15 scaffolds with an N50 length of 4,037,616 bp. The resulting GZL1 draft genome is 43,214,190 bp, with GC content of 58.19%. The completeness of GZL1 genome assembly is 99.30%. This study is the first report of the genome sequence of N. oryzae, which can facilitate future study of the genetic variation and pathogenic mechanism of this important fungal pathogen.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

First Report of Paramyrothecium foliicola Causing Stem Canker of Cucumber (Cucumis sativus L.) seedlings in China.

In April 2017, stem canker symptoms were observed on cucumber seedings grown in a greenhouse (0.1 ha) in Wuqing District, Tianjin(39°34' N; 117°07' E), China. Initially, the observed symptoms included small necrotic lesions of a light brown color on the stem base. These lesions subsequently spread and turned a darker brown. The leaves of the affected plants turned yellow and wilted. As the disease progressed the plants eventually died. Years of growing cucumbers and sufficient soil moisture in the greenhouse, might have led to a disease incidence of approximately 7%. Symptomatic tissue pieces were surface disinfested in 2% sodium hypochlorite for 60 s, rinsed three times in sterile water, and subsequently plated on potato dextrose agar (PDA) incubated at 25°C . At three days of incubation, mycelia appeared, turned into white and floccose isolated colonies around the excised tissue, and developed olivaceous green concentric rings of sporodochia in the following days. A total of 20 isolates with similar morphology were examined. Five single-spore isolates of isolates designated TJWQPF1-TJWQPF5 were obtained and maintained on PDA at 25°C. Hyaline, cylindrical conidiogenous cells measuring 9.53 to 16.51 × 1.51 to 2.49 µm (n=50) developed in whorls of three to six on terminal branches. Conidia were single-celled, hyaline, and rod-shaped with rounded ends. Conidia size averaged 5.07 - 7.15 × 1.13 - 2.32 µm (n=50). These characteristics are similar to the morphology of Paramyrothecium foliicola (Lombard et al. 2016). To further identify the isolate TJWQPF1, genomic DNA was extracted and the internal transcribed spacer (ITS, White et al. 1990), ß-tubulin (tub2, Glass & Donaldson 1995), RNA polymerase II largest subunit (rpb2, O'Donnell et al. 2007) and calmodulin (cmdA, Carbone & Kohn 1999; Groenewald et al. 2013) genes regions were amplified using the primer pairs ITS4/ITS5, Bt2a /Bt2b, RPB2-5F2 /RPB2-7cR, CAL-228F /CAL2Rd , respectively. All sequences were obtained and deposited in GenBank. BLAST searches of the NCBI database revealed that the ITS ( MW092223 ), tub2( MW110635 ) , rpb2 ( MW110637 ) and cmdA ( MW110636 ) sequences of the isolate TJWQPF1 were 100% identical to Paramyrothecium foliicola (GenBank accession numbers MT415351 and MT415352 for ITS sequences; MT415353 for tub2 sequences; MN398028-MN398043 for rpb2 sequences; MN593698- MN593713 for cmdA sequences). We also sequenced the other four single isolates and identified them as P. foliicola. Pathogenicity tests were conducted and repeated three times. Briefly, ten healthy 45-day-old cucumber seedlings (cultivar:Jinlv No.3) were inoculated with 100 µL of conidial suspension of P. foliicola (5×105 conidia per ml). Inoculum was applied to the stem with a syringe. Three healthy cucumber seedlings had 100 µL sterile water injected into the stem to serve as controls. All treated plants were incubated in a climate-controlled growth chamber at 25℃ (90% humidity, 12:12 h light:dark). Symptoms appeared on all inoculated plants after 7 days. In contrast, control seedlings exhibited no symptoms. The fungus was re-isolated from symptomatic tissues and re-identified to be P. foliicola, thereby fulfilling Koch's postulates. To our knowledge, this is the first known instance of P. foliicola inducing stem canker on cucumber plants in China. Stem canker caused by P. foliicola could pose a threat to cucumber production in China. Our results also provide a basis to monitor and manage this potential disease.

Genome Sequence Resource for Colletotrichum scovillei, the Cause of Anthracnose Disease of Chili.

Colletotrichum species cause anthracnose disease on the economically important spice crop chili. A total of 24 Colletotrichum species are known to infect chili and cause anthracnose. C. scovillei belongs to the C. acutatum species complex, and it shows greater aggressiveness than other species, particularly in the case of inoculation onto the nonwounded fruits of chili plants. The current work introduces an initial Illumina-Nanopore hybrid draft genome for C. scovillei TJNH1 together with the related annotations. Knowledge of this genome sequence provides an important reference genome of C. scovillei and will help further understand the pathogenic mechanism of C. scovillei to plant.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

First Report of Colletotrichum capsici Causing Anthracnose on Alocasia macrorrhizos in China.

Alocasia macrorrhizos (Linnaeus) G. Don is a perennial herb in the Araceae family. It is native to South Asia and the Asia-Pacific and has long been cultivated as it is an economically important medicinal and ornamental plant. During July 2012 and 2013, severe outbreaks of leaf spot and stem rot disease on this plant occurred in a greenhouse of Shunyi district, in Beijing, China (117°05'E, 40°13'N). The disease incidence was greater than 30%. The leaf spots first appeared as yellow dots. As lesions expanded, the symptoms were circular to subcircular, light brown lesions with darker brown edges, Around the lesions the leaf tissue was chlorotic causing the formation of a yellow halo (Suppl. Fig1). Initial symptoms on the stems were brown, round or fusiform spots . As the disease progressed, lesions enlarged and merged together. When humidity was high, black acervuli with grey brown cirrhus of conidia were rapidly produced in lesions. Infected plants eventually withered or collapsed from the stem rot (Suppl. Fig2). Infected tissues were surface-sterilized in 1% NaOCl for 1 min, washed three times with distilled water, and placed on potato dextrose agar (PDA). Colonies on PDA, growing at 25°C in darkness, showed grayish brown and grey brown conidial masses produced from acervuli with black seta (Suppl. Fig3). Acervuli (n=30) were dark brown to black and approximately round, 121 to 210 µm in diameter, averaging 166.5 µm (Suppl. Fig4). Setae (n=30) scattered in acervuli, black, septate, 94.4 to 128.4×3.4 to 4.7 µm, base inflated, and narrower toward the top (Suppl. Fig5). Conidiophores (n=50) were phialidic, hyaline, unicellular. Conidia (n=50) were hyaline, monospora, falcate, base obtuse, apices acute, and 20.5 to 24.7 ×2.8 to 3.4 µm (Suppl. Fig6). Six monoconidial isolates were made, and the morphological characteristics of the fungus were similar to those of Colletotrichum capsici (Syd.) Butler & Bisby (Mordue, 1971). In the greenhouse (25 to 30 °C, relative humidity 98%), pathogenicity tests were conducted by spraying a 106 spores /mL suspension on leaves and stems of 10 healthy potted A. macrorrhizos plants (3-year-old). A control was included that consisted of ten plants sprayed with sterile distilled water. All treated plants were covered with a plastic bag and removed 48 h later. After 12 days, all inoculated leaves and stems appeared with typical Anthracnose symptoms, whereas control plants remained healthy. The fungus was reisolated from diseased tissues, fulfilling Koch´s postulates. The ITS region of a representative isolate was amplified and sequenced using the primers ITS1/ITS4 (White et al. 1990).The obtained ITS sequence (GenBank Accession No. KJ018793.1) showed 100% similarity to Colletotrichum capsici (Accession No. HQ271469.1 and DQ454016.1). Colletotrichum capsici is synonymous to Colletotrichum truncatum. Colletotrichum capsici is a major phytopathogen with a broad host range which causes anthracnose disease. The first report of C. capsici as a pathogen of Alocasia macrorrhizos was reported in India in 1979 (Mathur, 1979). To our knowledge, this is the first record of C. capsici causing anthracnose on A. macrorrhizos in China.

Development and Implementation of an End-Effector Upper Limb Rehabilitation Robot for Hemiplegic Patients with Line and Circle Tracking Training.

Numerous robots have been widely used to deliver rehabilitative training for hemiplegic patients to improve their functional ability. Because of the complexity and diversity of upper limb motion, customization of training patterns is one key factor during upper limb rehabilitation training. Most of the current rehabilitation robots cannot intelligently provide adaptive training parameters, and they have not been widely used in clinical rehabilitation. This article proposes a new end-effector upper limb rehabilitation robot, which is a two-link robotic arm with two active degrees of freedom. This work investigated the kinematics and dynamics of the robot system, the control system, and the realization of different rehabilitation therapies. We also explored the influence of constraint in rehabilitation therapies on interaction force and muscle activation. The deviation of the trajectory of the end effector and the required trajectory was less than 1 mm during the tasks, which demonstrated the movement accuracy of the robot. Besides, results also demonstrated the constraint exerted by the robot provided benefits for hemiplegic patients by changing muscle activation in the way similar to the movement pattern of the healthy subjects, which indicated that the robot can improve the patient's functional ability by training the normal movement pattern.