First Report of black spot of Shine Muscat fruit caused by Cladosporium allicinum in China.

Shine muscat is a Vitis vinifera hybrid (Akitsu-21 × Hakunan), that has emerged as a popular table grape cultivar in China. In recent years, shine muscat has been widely cultivated with 66,667 ha being cultivated in 2021. In November 2021, symptoms of fruit spot were observed on shine muscat during the storage at 0~3℃ and 85%~90% RH, while stored at National Agricultural Product Preservation Engineering Technology Research Center, in Tianjin (N 116°20', E 39°09'), China. The incidence of this disease was about 35%. Affected grape berries initially had small brown spots. The spots on the fruit expanded to an ellipse or circular sunken area with a black center. The central peel of the diseased spots were ruptured and collapsed. The diseased fruits eventually fell off the vine. To isolate the pathogen, grape peels with typical symptoms were cut into small pieces, sterilized with 75% ethanol for 45-sec, rinsed with sterilized distilled water three times, and then transferred onto potato dextrose agar (PDA) medium.The plates were incubated at 25°C in the dark. After 10 days, 26 single spore isolates with similar morphology were obtained from 30 symptomatic grape berries. Fungal colonies were grayish brown, with abundant conidia on the obverse-side on PDA. Conidiophores were cylindrical, straight with unbranched, solitary or clustered, elongation at the tip and ranged in size from 3.2-6.8 × 35.6-150.9 µm (n=50). Conidia were grew in chains, ovoid, aseptate, and 2.2-6.0 × 8.3-16.8 µm (n=50). The morphological characteristics were consistent with Cladosporium allicinum (Bensch et al. 2012). Molecular data were also used to support the microscopic identification by extracting genomic DNA from 26 isolates using a Plant Genomic DNA kit (Tiangen, China). Amplicons were generated for the internal transcribed spacer (ITS), translation elongation factor 1-alpha(tef1-α), and actin (act) using the following primers ITS1/ITS4, EF1-728F/ EF1-986R and ACT-512F/ ACT-783R, respectively (Bensch et al. 2012). Blast analysis showed that three amplified fragments of 26 isolates were highly similar to C. allicinum, with 98.96~100% sequence identity with Cladosporium allicinum accessions in GenBank (ITS, OK661041; tef1-α, MF473332; act, LN834537). Three amplified fragments of representative isolate YG03 were deposited in GenBank with accession nos. OP799670 for ITS, OP888001 for tef1-α and OP887999 for act, respectively. Neighbor-joining trees based on concatenated sequences of three genes were constructed using MEGA5.2. The results showed that the strain YG03 from shine muscat was closely related to C. allicinum. Pathogenicity tests of 26 isolates were performed on healthy shine muscat berries using pin pricks and a humidor. In each wound, 5 µL of conidial suspension (1×106 conidia/mL) and sterile distilled water were inoculated on 30 berries, and maintained in a dark incubator at 25°C, 90% relative humidity. Each treatment was repeated twice. After 10 days, the wounded berries inoculated with the spore suspension showed dark brown spots, similar to the original diseased fruits, while no symptoms were observed on the control treament. Pathogen re-isolated from inoculated fruits were identical to the original strains on colony and microscopic morphology, and identified to Cladosporium allicinum based on act gene by molecular method, thereby fulfilling Koch's postulates. C.allicinum has been reported causing leaf spot on 11 host plants around the world (Bensch et al. 2012, 2015; Quaedvlieg et al. 2014; Jurisoo et al. 2019). To our knowledge, this is the first report of C. allicinum causing black spot on fruit of Vitis vinifera worldwide. The identification of this disease could establish a foundation for developing management strategies to reduce losses in storage period.

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.

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.

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.

First Report of Corynspora cassiicola Causing Leaf Spot on Jasminum sambac in China.

Jasminum sambac (Linnaeus) Aiton belonging to Oleaceae family, has been cultivated in China over 1500 years. It is an important tea crop in South China, and popular ornamental plant in North China. In June 2015, Corynespora leaf spot on Jasminum sambac was first observed in Caozhuang in Tianjin(E: 117°20', N: 39°13'). In a series of investigations, the disease incidence of Corynespora leaf spot on Jasminum sambac in Tianjin was found to be as high as 20%. The disease was also found in flower planting greenhouses in Beijing and Hebei region. The initial symptoms appeared as small circular spot with gray white in the center and dark brown in the edge. The lesions expanded rapidly to 5-10 mm diameter, surrounded by a yellow halo. The affected leaves became covered with the circular spots and defoliated eventually. Fifty-six leaf samples were collected from 20 greenhouses, then directly examined and disease tissues set aside for isolation. Conidiophores were either solitary or in clusters, straight or curved and un-branched, with 3-10 septate, pale to dark brown in color. Those were measured 80.6 to 234.8 µm long and 4.5 to 8.0 µm wide in size and were formed with 0-8 successive cylindrical proliferation (n = 50). Conidia borne from conidiophore, were single or in chains, obclavate to cylindrical, smooth. The average size of 50 conidia was 43.6 to 130.7 µm long×6.4 to 13.5 µm wide, with 4 to 8 pseudosepta (n = 50). The infected leaves were cut into small pieces, sterilized with 75% ethanol, rinsed in sterilized distilled water and then plated on potato dextrose agar (PDA). The isolated plates were incubated at 25℃ for 5 days. Thrity-five pure culture isolates were obtained from diseased leaves. The colonies were pale brown on the front and dark brown on the underneath, with thickly aerial mycelia. Morphological characteristics of the pathogen from the infected leaves and purified strains matched descriptions of Corynesora cassicola (Ellis and Holliday 1971). To confirm the pathogenicity, conidial suspensions (106 conidia mL-1 ) of 35 isolated strains were sprayed on the healthy plants at the seedling stage of Jasminum sambac respectively, 5 plants per strains. 10 healthy plants were sprayed only with sterilized water as control. Conidial suspensions were prepared by washing conidia from the pure C. cassiicola cultures. Conidial suspensions were adjusted to a concentration of 1 × 106 conidia/mL with the hemocytometer. All plants were incubated in artificial climate-room at 90% relative humidity, 25±2℃. After 7 days, small circular spots, similar to the field samples were observed on all plants inoculated with the pathogen. By comparison, control plants displayed no symptoms. The same pathogens were re-isolated from the symptomatic leaves fulfilling Koch's postulates. For molecular identification, rDNA-ITS and two housekeeping genes (TUB2 and EF-1α) gene were amplified and sequenced (Zhu et al. 2020). The sequences of three high pathogenicity isolats showed 99% homology to C.cassiicola (MK571399) with 464 bp for rDNA-ITS, 100% homology to C.cassiicola (MN512639) with 469 bp for TUB2 gene, 98.61% homology to C.cassiicola (MH263735) with 287 bp for EF-1α gene. And all 9 sequences were submitted to GenBank (GenBank accession No. MT334575-77 for rDNA-ITS, MT886387-89 for TUB2, and MT886390-92 for EF-1α).The molecular identification and morphological characteristics showed that the isolated strains from Jasminum sambac were identified as C.cassiicola. C. cassiicola has an extensive host range, covering 397 host plants (Farr and Rossman 2020). Recently, C.cassiicola has been reported on Jasminum mesnyi of Oleaceaein China (Zhang et al. 2018). To our knowledge, this is the first report of leaf spot caused by C.cassiicola on Jasminum sambac in China and also worlwide.

Quantification of Viable Cells of Pseudomonas syringae pv. tomato in Tomato Seed Using Propidium Monoazide and a Real-Time PCR Assay.

Pseudomonas syringae pv. tomato is a seedborne pathogen that causes bacterial speck disease in tomato. P. syringae pv. tomato is typically detected in tomato seed using quantitative real-time PCR (qPCR) but the inability of qPCR to distinguish between viable and nonviable cells might lead to an overestimation of viable P. syringae pv. tomato cells. In the present study, a strategy involving a propidium monoazide (PMA) pretreatment followed by a qPCR (PMA-qPCR) assay was developed for quantifying viable P. syringae pv. tomato cells in contaminated tomato seed. PMA could selectively bind to the chromosomal DNA of dead bacterial cells and, therefore, block DNA amplification of qPCR. The primer pair Pst3F/Pst3R was designed based on gene hrpZ to specifically amplify and quantify P. syringae pv. tomato by qPCR. The PMA pretreatment protocol was optimized for selectively detecting viable P. syringae pv. tomato cells, and the optimal PMA concentration and light exposure time were 10 µmol liter-1 and 10 min, respectively. In the sensitivity test, the detection limit of PMA-qPCR for detecting viable cells in bacterial suspension and artificially contaminated tomato seed was 102 CFU ml-1 and 11.86 CFU g-1, respectively. For naturally contaminated tomato seed, viable P. syringae pv. tomato cells were quantified in 6 of the 19 samples, with infestation levels of approximately 102 to 104 CFU g-1. The results indicated that the PMA-qPCR assay is a suitable tool for quantifying viable P. syringae pv. tomato cells in tomato seed, which could be useful for avoiding the potential risks of primary inoculum sources from contaminated seed.