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.

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.

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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), β-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.

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, β-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.

During September 2010, Abutilon megapotamicum plants with darkbrown concentric spots on leaves were observed in a commercial glasshouse located in Beijing, China. This study was carried out to identify the causal agent of this disease based on Koch's postulates and morphological characteristics. Pathogenicity tests in the glasshouse showed that Myrothecium roridum Tode ex Fr. caused the leaf spot on A. megapotamicum plants, which were the same as those observed in naturally infected plants in the field. Moreover, to confirm the pathogen to species, the rDNA internal transcribed spacer (ITS) of isolate was PCR-amplified using ITS1 and ITS4 primer pairs and sequenced. DNA analysis revealed a 100% species identity index for M. roridum. To the best of our knowledge, this is the first report of M. roridum on A. megapotamicum in China.

Zantedeschia aethiopica (L.) Spreng. (calla lily), belonging to family Araceae, is a popular ornamental plant in China. In the summer of 2010, leaves of calla lily with typical symptoms of necrotic lesions were observed in a commercial glasshouse in Beijing, China (116°20′ E, 39°44′ N). The initial symptoms were circular to subcircular, 1 to 3 mm, and dark brown lesions on the leaf lamina. Under high humidity, lesions expanded rapidly to 5 to 10 mm with distinct concentric zones and produced black sporodochia, especially on the backs of leaves. Later, the infected leaves were developing a combination of leaf lesions, yellowing, and falling off; as a result, the aesthetic value of the plant was significantly impacted. Leaf samples were used in pathogen isolation. Symptomatic leaf tissues were cut into small pieces and surface sterilized with 70% ethanol for 30 s and then in 0.1% mercuric chloride solution for 1 to 3 min. After being washed in sterile distilled water three times, the pieces were plated on potato dextrose agar (PDA) and incubated at 25°C in darkness for 7 days (5). Initial colonies of isolates were white, floccose mycelium and developed dark green to black concentric rings that were sporodochia bearing viscid spore masses after incubating 5 days. Conidiophores branched repeatedly. Conidiogenous cells were hyaline, clavate, and 10.0 to 16.0 × 1.4 to 2.0 μm. Conidia were hyaline, cylindrical, both rounded ends, and 6.0 to 8.2 × 1.9 to 2.4 μm. Morphological characteristics of the fungus were consistent with the description of Myrothecium roridum Tode ex Fr. (3,4). To confirm the pathogenicity, three healthy plants of calla lily were inoculated with a conidial suspension (1 × 106 conidia per ml) brushed from a 7-day-old culture of the fungus. Control plants were sprayed with sterile water. The inoculated plants were individual with clear plastic bags and placed in a glass cabinet at 25°C. After 7 days, all inoculated leaves developed symptoms similar to the original samples, but control plants remained disease free. Re-isolation and identification confirmed Koch's postulates. For molecular identification, genomic DNA of a representative isolate (MTL07081001) was extracted by modified CTAB method (1), and the rDNA-ITS region was amplified by using primers ITS1 (5-TCCGTAGGTGAACCTGCGG-3) and ITS4 (5-TCCTCCGCTTATTGATATGC-3). The 465-bp amplicon (GenBank Accession No. KF761293) was 100% identity to the sequence of M. roridum (JF724158.1) from GenBank. M. roridum has an extensive host range, covering 294 host plants (2). To our knowledge, this is the first record of leaf spot caused by M. roridum on calla lily in China. References: (1) F. M. Ausubel et al. Current Protocols in Molecular Biology. John Wiley & Sons Inc, New York, 1994. (2) D. F. Farr and A. Y. Rossman, Fungal Databases. Syst. Mycol. Microbiol. Lab., ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , October 2013. (3) M. T. Mmbaga et al. Plant Dis. 94:1266, 2010. (4) Y. X. Zhang et al. Plant Dis. 95:1030, 2011. (5) L. Zhu et al. J. Phytopathol. 161:59, 2013.

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.