First report of Stemphylium lycopersici causing leaf spot on Polygonatum kingianum in China

Polygonatum kingianum, a member of the Liliaceae, is valued in traditional medicine and as a vegetable food crop. In July 2019, more than 50% of P. kingianum growth was suppressed in several field nurseries in Simao, Mojiang, Jingdong and Lancang County, Puer City, China. At the early stage of infection, symptoms manifested as a small circular brown spot. As the lesion matured, the spot gradually enlarged, forming an oval to irregular lesion with reddish-brown and dark green borders. In serious cases, the leaves were withered, and became brittle with cracks. The infected plants were collected from six major fields. The tissues of diseased leaves were soaked in 75% ethanol for 10 s, 0.1% mercuric chloride for 2 min, rinsed with sterilized water, and placed on potato dextrose agar (PDA) at 25℃ for 7 days. On PDA, four strains were isolated, and the colony was gray to dark yellowish-brown, flocculent, regular with concentric growth rings. Strain PKLS06 produced a dark red to brown pigment in the agar medium. On lesions, the conidiophores were solitary or in fascicles, straight or slightly curved, brown, with a conical apex, with three to five septa. The conidiogenous cells were pale-brown and swollen at the apex. On PDA, spores were solitary, oblong, bluntly rounded or sometimes with a point at the apex, with two to five transverse septa and one to two longitudinal septa with contractions at the main transverse septum. Morphological characteristics were consistent with published descriptions of Stemphylium lycopersici (Kee et al. 2017; Xie et al. 2018). For molecular identification, rDNA internal transcribed spacer (ITS) and the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene were amplified and sequenced (ITS accessions: MW243098, MW243099, MW243100, MW243101; gpd accessions: MW246803, MW246804, MW246805, MW246806) using published primers (White et al. 1990; Câmara et al. 2002). A phylogenetic tree was developed by Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian inference (BI). These four isolates fall into the S. lycopersici clade with strong support and all isolates were distinguished clearly from other species. Pathogenicity tests were performed using these four isolates. Each isolate was cultured on PDA and shake-cultured in V-8 juice broth (Nasehi et al. 2014). Conidia were resuspended in sterilized water (1×106 conidia/mL) and inoculated on intact leaves with injury of 1-year-old P. kingianum. The plants were incubated at 25℃ with a 12 h photoperiod and 90% humidity. A small spot began to appear after 3 days, and symptoms were similar to the those observed in the nursery after 10 days. Interestingly, the pathogenicity of strain PKLS06 was relatively weaker. Control plants treated with sterile water showed no disease symptoms. Re-isolated strains had the same morphological characteristics and the same ITS and gpd sequences as the original isolates, thus fulfilling Koch’s postulates. S. lycopersici, an important pathogen, is widely distributed, and can cause a variety of plant diseases. It is noteworthy that the disease was observed on a plant in the Liliaceae, expanding the host range of S. lycopersici, which previously was reported to primarily infect plants in the Solanaceae. Based on the results presented above, P. kingianum is a new host plant of S. lycopersici in China. This disease is a threat for nursery production of P. kingianum, leading to a reduction in yields and economic losses. References Kee, Y. J., et al. 2017. Plant Disease 102 (2): 445–446 Xie, X. W., et al. 2019. Canadian Journal of Plant Pathology-Revue Canadienne De Phytopathologie 41 (1): 124–128 White, T. J., et al. 1990. PCR Protocols: A Guide to Methods and Applications PCR Protocols: A Guide to Methods and Applications 18: 315–322 Câmara M. P. S., et al. 2002. Mycologia 94 (4): 660–672 Nasehi A., et al. 2014. Archives of Phytopathology & Plant Protection, 47 (14): 1658-1665.

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-wide identification and functional analysis of the ERF2 gene family in response to disease resistance against Stemphylium lycopersici in tomato

Background APETALA2/ethylene responsive factor (AP2/ERF) transcription factors are a plant-specific family of transcription factors and one of the largest families of transcription factors. Ethylene response factors (ERF) regulate plant growth, development, and responses to biotic and abiotic stress. In a previous study, the ERF2 gene was significantly upregulated in both resistant and susceptible tomato cultivars in response to Stemphylium lycopersici. The main purpose of this study was to systematically analyze the ERF family and to explore the mechanism of ERF2 in tomato plants resisting pathogen infection by the Virus-induced Gene Silencing technique. Results In this experiment, 134 ERF genes were explored and subjected to bioinformatic analysis and divided into twelve groups. The spatiotemporal expression characteristics of ERF transcription factor gene family in tomato were diverse. Combined with RNA-seq, we found that the expression of 18 ERF transcription factors increased after inoculation with S. lycopersici. In ERF2-silenced plants, the susceptible phenotype was observed after inoculation with S. lycopersici. The hypersensitive response and ROS production were decreased in the ERF2-silenced plants. Physiological analyses showed that the superoxide dismutase, peroxidase and catalase activities were lower in ERF2-silenced plants than in control plants, and the SA and JA contents were lower in ERF2-silenced plants than in control plants after inoculation with S. lycopersici. Furthermore, the results indicated that ERF2 may directly or indirectly regulate Pto, PR1b1 and PR-P2 expression and enhance tomato resistance. Conclusions In this study, we identified and analyzed members of the tomato ERF family by bioinformatics methods and classified, described and analyzed these genes. Subsequently, we used VIGS technology to significantly reduce the expression of ERF2 in tomatoes. The results showed that ERF2 had a positive effect on tomato resistance to S. lycopersici. Interestingly, ERF2 played a key role in multiple SA, JA and ROS signaling pathways to confer resistance to invasion by S. lycopersici. In addition, ERF2 may directly or indirectly regulate Pto, PR1b1 and PR-P2 expression and enhance tomato resistance to S. lycopersici. In summary, this study provides gene resources for breeding for disease resistance in tomato.