Confirmation of Stemphylium vesicarium, the causal agent of brown spot of tomato in Germany

The tomato is a major crop worldwide and is one of the five most consumed vegetables in Germany. Stemphylium species including S. eturmiunum, S. gracilariae, S. lycii, S. lycopersici, S. rombundicum, S. simmonsii, S. solani, and S. vesicarium have been identified as tomato pathogens in various countries. In Germany, multiple instances of S. lycopersici and S. solani affecting tomato plants have been documented; however, only one incident of S. vesicarium has been reported in 1972. S. vesicarium is known to cause yellowish-brown spots on tomato leaves, which can ultimately lead to defoliation and reduced fruit yield. Therefore, it is crucial to identify the S. vesicarium that cause this disease accurately. In this study, S. vesicarium was isolated from necrotic tomato plants in organic farms located in northern and southern regions of Germany. Single spore isolates were generated and identified as S. vesicarium based on morphological characteristic and molecular analyses using nucleotide sequences of the internal transcribed spacer (ITS) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Koch´s postulates were fulfilled and revealed that S. vesicarium is the causal agent of brown spot on the samples adding a new account of the species.


Introduction
Tomatoes are one of the five most valuable vegetable crops produced and consumed worldwide (FAO 2021) and in Germany. According to the Federal Information Centre for Agriculture (BZL), the number of tomatoes consumed by Germans in 2018/19, including processed tomatoes, reached 2.26 million tons. A majority of German tomatoes are grown in greenhouses and are susceptible to a variety of diseases that can develop during cultivation, production, transportation or storage (Kalloo 1991;Panno et al. 2021;Singh et al. 2017; Thole et al. 2021). Foliar tomato diseases can be caused by many different pathogens. For instance, the disease early bight is caused by Alternaria solani, "leaf mold" by Fulvia fulva, "powdery mildew" by Erysiphe neolycopersici, and "late blight" by the oomycete Phytophthora infestans (Gabler et al. 1990;Hsiao et al. 2022;Leiminger et al. 2014;Meyer and Gärber 2021;Möller et al. 2009). To that, certain species belonging to the Stemphylium genus have the ability to infect tomato plants.
Stemphylium Wallr. is a genus of filamentous ascomycetes that includes endophytic, saprophytic, and plant pathogenic species, several of which cause diseases in agricultural crops (McNeill et al. 2012;Simmons 2001;Simmons 1969). S. vesicarium is a widespread fungal pathogen that can infect over fifteen different host genera worldwide. Recently, it has been reported to cause various diseases on different host plants, such as wilting and root rotting of radish sprouts, gray leaf spot in chilli peppers, and brown spot of pear disease (Belisario et al. 2008;Llorente and Montesinos 2002;Sharifi et al. 2021;Vitale et al. 2021).
In Germany, S. vesicarium was originally described from garlic (Wallroth 1833) and has caused serious economic losses in crops such as garlic, asparagus, and pears (Graf et al. 2016;Llorente and Montesinos 2002). Purple spot disease is a problem for asparagus-growing areas. During autumn, it causes premature defoliation of asparagus ferns and reduces 1 3 photosynthetic capacity by up to 52% (Graf et al. 2016). On tomato plants, symptoms of S. vesicarium infection begin a brown or gray spot surrounded by a yellow halo, which eventually progresses to form necrotic lesions with a brown center and dark brown borders. Affected leaves turn chlorotic, become dry, crack, and fall off, thus resulting in a loss of tomato yield (Blancard et al. 2012). Approximately eight Stemphylium species are associated with diseases of tomato including: S. eturmiunum, S. gracilariae, S. lycii, S. lycopersici, S. rombundicum, S. simmonsii, S. solani, and S. vesicarium (Câmara et al. 2002;Inderbitzin et al. 2009;Nasehi et al. 2012;Woudenberg et al. 2017). Among others, S. lycopersici and S. solani are the most common species causing gray leaf spot and black spot, respectively. In some cases, symptoms caused by S. vesicarium in tomato plants can be confused with those caused by Alternaria species which look very similar in the field. Therefore, it is often difficult to make a definitive diagnosis of S. vesicarium infection based solely on visual observations (Fernández and Rivera-Vargas 2008;Huang and Tsai 2017). There have been reports of tomato plants infected by S. vesicarium in various countries, including Algeria, Australia, and the USA. This suggests that S. vesicarium is a pathogen that can be found in different parts of the world and can potentially cause damage to tomato crops (Bessadat et al. 2022;Marin-Felix et al. 2019;Woudenberg et al. 2017).
During an extended survey on Fulvia fulva pathotypes associated with organically cultivated tomato in Germany 2020 and 2021, atypical necroses were observed on tomato leaves of different cultivars and breeding lines at two locations in Germany. The aim of the present study was to identify and confirm S. vesicarium as the causal agent of these symptoms. Molecular and taxonomic analyses were used to identify the pathogenic fungus. Koch's postulates were fulfilled by means of infection and re-isolation of the pathogen.

Isolation and maintenance
In 2020 and 2021, leaves with symptoms of brown spots and/or dark necrotic lesions were collected from organically cultivated tomato plants at two locations in Germany (Table 1). At each location, at least five samples were collected from each of the four tomato cultivars. Symptomatic tomato leaves were immediately inspected microscopically for the presence of spores to indicate a fungal infection. Necrotic areas were surface disinfected by immersion in 0.5% sodium hypochlorite solution for 1 min followed by two washing steps in sterile water for 2 min each. The disinfected tissues were dried in a laminar flow cabinet and used for fungal isolation. To verify surface disinfection was successful, both sides of the dried leaf pieces were pressed onto potato dextrose agar (PDA). Leaf tissues were sectioned and 0.5 cm 2 pieces were placed in petri dishes on PDA culture media. Plates were incubated at 25 °C in the dark and inspected regularly for mycelial growth. Once the hypha emerged from the leaf tissue, the tips were transferred onto fresh PDA medium and incubated until conidia were produced. To obtain single spore cultures, conidia were individually transferred to fresh PDA and maintained at 25 °C. Identification of the single spore isolates were performed based on morphological characteristics and molecular data. One isolate per location was deposited in the culture collection of the Institute for Plant Protection in Horticulture and Urban Green (JKI) (GFP-22-007-009) and the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSM 114463-114465), Braunschweig, Germany.

Morphological characterization
After 10 and 14 days cultivation, the growth rates and characters of fungal colonies were measured and described. Morphological characteristics were examined using a light microscope (Axio Imager.A1 equipped with an AxioCam MRc5 camera, Zeiss, Germany). The size and shape of conidia (n = 50) and conidiophores (n = 50), as well as the position of the basal septum, were analyzed morphologically. Color descriptions were determined by comparison to a color chart (Rayner 1970). Measurements were made and images were taken with the calibrated ZEN Blue Edition software rel. 3.1 (Zeiss, Germany). The images were edited using Adobe Photoshop CS6 software (Adobe Systems, USA).

Phylogenetic analyses
Forward and reverse sequences were assembled and edited with Geneious Pro 5.

Pathogenicity tests
To fulfill Koch's postulates and test the susceptibility of tomato toward S. vesicarium pathogenicity tests were performed with the tomato cultivars "Moneymaker," "Belafonte," and three additional breeding lines. A spore suspension was prepared from single spore cultures grown on PDA for 10 to 14 days. The cultures were submersed with sterile distilled water containing one droplet of 0.1% Tween 20 and spores were removed from the mycelium with a sterile glass rod. The spore suspension was filtered through cheesecloth, the concentration was determined using a hemocytometer and adjusted to 1 × 10 6 conidia/ml. Five tomato plants per cultivar/breeding line at the 4-to 5-true-leaf stage (21 days old) were sprayed with approximately 40 ml of the spore suspension on the adaxial side of the leaves. Control plants were treated in the same way with sterile distilled water. Plants were grown in rectangular plastic containers under the following greenhouse conditions: 99% relative humidity, enclosed chamber for 72 h, at 22 ± 3 °C, 16 h light/8 h dark period. Afterward they were cultivated in open plastic containers for 21 days and evaluated regularly for visible disease symptoms. A scale 0-4 was used according to the disease severity symptoms of the plants; 0 = no infection, 1 = 25%, 2 = 50%, 3 = 75%, and 4 = 100% necrosis of the adaxial side of leaves. The final disease assessment was conducted 21 days after inoculation. To verify that developing symptoms were caused by S. vesicarium re-isolation of the pathogen from symptomatic leaves of each cultivar was performed as described above.

Morphological and molecular identification/ characterization of S. vesicarium
A colony of S. vesicarium developed after 7 days of incubation on PDA at 25 °C in the dark (Fig. 1c, d). The colony exhibited dense mycelium, 50-75 mm in diameter, buff-yellow with regular margins, and a brownish olive to buffy brown center. Conidiophores were straight or occasionally branched, each with one or two swollen apices and two to 11 septae, and were of short to moderate size between 30-160 × 4-10 μm. Conidia were medium to deep brownish, oblong, ellipsoid to muriform, with one to three transverse segments and one to four longitudinal septae per transverse segment. The conidia were each constricted at one to three of the major transverse septae. Conidia were 20-40 × 15-27 μm, with mean length/width ratio of 1.2 ± 0.3 μm. Sexual morphs were not observed. A BLAST search on the ITS sequence of our S. vesicarium isolates indicated 100% similarity and 100% query cover with a sequence from the ex-type isolate of S. tomatonis CBS 109844 (GenBank: KU850572)-a synonym of S. vesicarium (Woudenberg et al. 2017). Likewise, 100% identity and 100% query cover to S. vesicarium CBS 109844 (GenBank: KU850719) was returned for GAPDH sequence. The combined genes phylogenetic analyses indicated that S. vesicarium clustered with other S. vesicarium isolates over the world, with a well-supported bootstrapping value (BS) of 100%. The topology of the phylogenetic tree (Fig. 2) obtained in this study was consistent with previously published trees without supported conflicts (Brahmanage et al. 2019;Inderbitzin et al. 2009;Woudenberg et al. 2017).

Pathogenicity tests
After 4 to 7 days of cultivation, leaf spot symptoms appeared on the inoculated tomato plants and the lesions increased progressively toward necrosis with time. Symptom progression followed the expected pattern starting with dark brown lesions surrounded by yellowish halos, and later advancing to diffuse leaf blight (Fig. 1a, b). Two isolates JKI-GFP-22-007 and JKI-GFP-22-009 were classified as highly virulent as indicated by a disease severity symptom score of 4, which signifies that the isolates produced necrotic leaf lesions that expanded to the entirety of the leaf in all inoculated plants. However, infection with isolate JKI-GFP-22-008 resulted in a disease severity symptom score of 2, signifying that the isolate caused only mild symptoms and the small necrotic lesions did not expand. S. vesicarium was subsequently re-isolated from all inspected lesions and identified as the same species according to the morphological characteristics. No symptoms were observed on control leaves treated with sterile water and no mycelium was detected after surface disinfection of these leaves.

Discussion
The first report of S. vesicarium was found on garlic by Wallroth in Germany in 1833. However, neither the isolate or ex-epitype culture was deposited in a collection for verification. Identification of the fungus can be complex as the nomenclature for this pathogen has changed repeatedly over relatively long periods of time (see https:// www. index fungo rum. org). Based on molecular studies, S. vesicarium was initially considered identical to S. alfalfae and S. herbarum. Morphological examinations do not easily distinguish characteristics between species, but in recent years, a more extensive phylogenetic analysis has suggested that S. alfalfae and S. herbarum are synonymous with S. vesicarium (Brahmanage et al. 2019;Câmara et al. 2002;Inderbitzin et al. 2009;Köhl et al. 2009;Marin-Felix et al. 2019;Nasehi et al. 2014;Woudenberg et al. 2017). The findings of this study confirm the tomato plant pathogen collections as S. vesicarium by using a combination of ITS and GAPDH sequences that distinguish the fungi at the species level and morphological descriptions. Woudenberg et al. (2017) reported the synonymization of S. herbarum with S. vesicarium and identified S. vesicarium as the causal agent of tomato disease from Oberfranken in 1972. This incident represents the only known report of S. vesicarium infecting tomato plants in Germany. During our survey on F. fulva infecting organically grown tomato plants, we found atypical necrotic spots at several locations over two consecutive years. The pathogens isolated from symptomatic tissue were identified as S. vesicarium based on morphological characteristics and phylogenetic analysis. The virulence of the isolates in tomato was quantified using a disease severity symptom score and subsequently re-isolation was conducted to fulfill Koch's postulates. All three isolates caused brown to dark necrotic lesions on tomato leaves under greenhouse conditions comparable to those observed in the field.
As the original survey was on F. fulva pathotypes, tomato leaves with atypical symptoms were collected and presence of S. vesicarium was confirmed for these locations. Therefore, no conclusive statement can be made about the distribution and damage caused by S. vesicarium in Germany to date, especially as the symptoms closely resemble those of another fungal infection, e. g., caused by Alternaria spp. However, there is evidence that the pathogen seems to be Fig. 2 The RAxML phylogenetic tree based on combined ITS and GAPDH sequences from taxa of Stemphylium species that are associated with diseases of tomato plants.
Bootstrap support values equal to or greater than 70% are given above the branches. Ex-epitype, -type, -holotype, and -neotype isolates are indicated with ET, T, HT, and NT, respectively. Stemphylium vesicarium from this study are shown in blue. Isolate identifier, host and country are provided after each species name. The tree is rooted to Alternaria abundans and A. breviramosa widespread in the country since we have found it in two further locations in 2022 (data not shown). Additionally, it has the potential to cause disease in various tomato cultivars/ breeding lines. A comprehensive monitoring could provide information on the distribution and relevance of S. vesicarium for organic tomato production in Germany.
In recent years, there have been concerns about the increasing prevalence of Stemphylium spp. leaf spot disease in tomato crops in many parts of the world. The disease can cause significant yield loss if no control measures are taken, and there are limited options for effective control. Future studies are required to investigate the conditions influencing the development of disease in tomato plants, the availability of resistant tomato cultivars, the interactions between susceptible/resistant hosts, and the composition of phytotoxic metabolites.
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