Solanum lycopersicum (Solanaceae) is one of the most important and broadly grown vegetables in the world. Brazil is among the major producers of tomato worldwide (Vale et al. 2007). A large range of fungal and oomycete diseases affect tomato (Jones et al. 2014; Lopes and Ávila 2005), some of which are universally regarded as major threats to tomato production, such as early blight, caused by Alternaria spp., and late blight, caused by Phytophthora infestans. Others, such as Phoma rot, are considered as having less relevance. In Brazil, this disease is considered to be of secondary importance (Inouie-Nagata et al. 2016). Although better known for causing post-harvest disease of tomato fruits, Phoma (Didymellaceae) occasionally also affects stems and leaves in the field (Kimati et al. 2005; Robl et al. 2014).

In July of 2015 several plants grown in a greenhouse in the campus of the Universidade Federal de Viçosa (Viçosa, state of Minas Gerais, Brazil), were found to bear severe leaf blight symptoms (Figs. 1a, b). Lesions also appeared on stems, which were similar to those seen on leaves (Fig. 1c). All diseased plants died because of the disease. A coelomycete asexual morph was repeatedly found in association with the necrotic tissues.

Fig. 1
figure 1

Phoma destructiva. a. Early stages of infection of Phoma destructiva on a tomato leaf. b. Ibid on flowers. c. Necrotic spots on stem and on a young sprout. d. Tomato plant with severe blight 20 days after inoculation. e. Transversal section of a pycnidium showing a layer of conidiogenous cells. f. Conidia with and without septa. Bars: e = 8 µm and f = 20 µm

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Direct isolation of the fungus in pure culture was performed through aseptic transfer of spores from colonized tissue onto PDA medium with a sterile fine-pointed needle. A representative isolate was deposited in the culture collection of the Universidade Federal de Viçosa, (Accession number COAD 2069). A representative dried specimen of infected tomato tissue was deposited in the herbarium of the Universidade Federal de Vicosa (VIC 44080). Sections of selected fragments of infected leaves bearing fruiting bodies were mounted in lactoglycerol for observation under a light microscope (Olympus BX 51) fitted with an Olympus e-volt 330 digital camera. Biometric data were recorded based on at least 30 measurements of various structures.

In order to obtain representative fungal DNA, COAD 2069 was grown on PDA at 25 °C under a 12-h daily light regime for one week. DNA was extracted from approximately 40 mg of fungal mycelium using the Wizard Genomic DNA Purification Kit (Promega) following the protocol described by Pinho et al. (2012).

The nrDNA (ITS) and partial β-tubulin (TUB) sequences of COAD 2069 were amplified using the primer pairs ITS1/ITS4 (White et al. 1990) and T1/Bt2b, (Glass and Donaldson 1995; O’Donnell and Cigelnik 1997), following the protocols described by Pinho et al. (2012) and Sung et al. (2007). Amplification of ITS and β-tubulin, produced sequences of approximately 540 and 567 bp, respectively. The nucleotide sequences were edited with the DNA Dragon software (Hepperle 2011) and deposited in GenBank under accession numbers MF 426960 for ITS and MF448543 for β-tubulin. Additional ITS and β-tubulin sequences used in this study were retrieved from GenBank (Table 1).

Table 1 GenBank accession numbers of Phoma spp. DNA sequences used in the phylogenetic analysis
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ITS and TUB consensus sequences were compared with others deposited in the GenBank database using the MegaBLAST program. The most similar sequences were aligned using MUSCLE (Edgar 2004) and built in MEGA v.5 (Tamura et al. 2011). All of the ambiguously aligned regions within the dataset were excluded from the analyses. Gaps (insertions/deletions) were treated as missing data. Bayesian inference (BI) analyses employing a Markov Chain Monte Carlo method were performed with all sequences, first with each gene separately and then with the concatenated sequences (ITS and β-tubulin).

Before launching the BI, the best nucleotide substitution models were determined for each gene with MrMODELTEST 2.3 (Posada and Buckley 2004). Once the likelihood scores were calculated, the models were selected according to the Akaike Information Criterion (AIC). The SYM + I + G model of evolution was used for ITS, whereas GTR + I + G was used for β-tubulin. The phylogenetic analysis of the concatenated sequences was performed on the CIPRES web portal (Miller et al. 2010) using MrBayes v.3.1.1 (Ronquist and Heulsenbeck 2003). The other phylogenetic analyses were conducted as described by Pinho et al. (2012). Sequences derived from this study were deposited in GenBank (http://www.ncbi.nlm.nih.gov/genbank) (Table 1), and the alignments and trees in TreeBASE (www.treebase.org) under entry number S21657.

A pathogenicity test was conducted including thirteen tomato varieties, namely: Forty, Ikram, Paronset, Platinum, Fusion, Santa Clara Siluet, Aguamiel, Caribe, Serato, Predador, Gladiador, Dominador and Vento. All varieties carry the Mi allele that confers resistance to root knot nematode (Meloidogyne spp.). COAD 2069 was grown on PDA medium at 25 °C for 7 days under a 12-h daily light regime and a conidial suspension (1 × 106 conidia/mL) was prepared for inoculation. Plants were sprayed with this suspension until runoff and kept for 2 days in a dew chamber at 25 ± 3 °C and later transferred to a greenhouse. In addition, two plants were sprayed with sterile water and kept under the same conditions to serve as controls.

Phoma destructiva var. destructiva Plowr., Gard. Chron. II 16: 621. 1881.

Disease: On leaves, stems and flowers, starting as small black dots (1 to 2 mm diameter) that became circular to somewhat irregular, slightly depressing the plant tissues and forming zonate necrotic areas, where numerous fruiting bodies were formed. Lesions coalesced with age and led to entire blight of leaves, flowers and stems.

Morphology: Internal mycelium indistinct; external mycelium absent; pycnidia immersed globose to irregular, 60–150 × 70–180 μm, walls of thick textura angularis, ostiolate, brown, smooth; conidiogenous cells enteroblastic, ampulliform to doliiform, 4–6 × 3.5–8, hyaline, smooth; conidia oval to ellipsoidal, 4.5–8 × 2.5–3 μm, mainly aseptate but larger 1-septate consistently produced, biguttulate, hyaline, smooth.

In culture: On PDA and PCA, fast-growing (5.5–7.5 cm diameter after 7 days), edge entire, slightly convex, aerial mycelium cottony centrally, followed by an area of sparse mycelia, centrally pale olivaceous green alternating with dark mouse grey, periphery composed of immersed mycelium, diurnal zonation present; slightly humid centrally, olivaceous black reverse; sporulation abundant on PCA.

Typical leaf blight symptoms developed on leaves, flowers and stems of all ten tomato varieties five days after inoculation. The fungus was isolated from diseased tissues of inoculated plants, producing typical P. destructiva colonies, confirming the pathogenicity of COAD 2069 to a range of tomato cultivars (Fig. 1). The morphological features of the fungus isolated from diseased tissue were typical of Phoma destructiva (Table 2) and this identification was strongly supported by phylogenetic analysis of concatenated ITS and TUB gene sequences (Fig. 2).

Table 2 Morphology of Phoma species recorded on Lycopersicum esculentum worldwide
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Fig. 2
figure 2

Phylogenetic study of Phoma destructiva. Tree inferred from Bayesian analysis based on concatenated ITS and TUB sequences. Species from Brazil are shown in bold face. Bayesian posterior probabilities are indicated at the nodes. The tree was rooted with Stagonosporopsis actaeae (isolate CBS 105.96 and CBS 106.96)

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Seven species of Phoma have been reported causing diseases on tomato in Brazil (Chen et al. 2015; Farr and Rossman 2016), including Phoma destructiva, which has been recorded causing fruit rot only once (Batista and Alves 1981). Nevertheless, that record is incomplete and obscure not supported by any morphological description of the fungus nor deposit of voucher specimens in herbaria or culture collections.

Based on morphology, P. destructiva has been separated into two varieties P. destructiva var. destructiva and P. destructiva var. diversispora differing in conidial size and septation (De Gruyter et al. 2002). Phoma destructiva var. destructiva is aseptate and has been reported as a common tomato and sweet pepper pathogen, causing fruit rot and small black spots on leaves and fruits in the tropics. Phoma destructiva var. diversispora is morphologically distinct from var. destructiva by producing larger one-septate conidia - a feature found in members of the section Phyllostictioides. (Aveskamp et al. 2010), and is considered to be restricted to Europe (De Gruyter et al. 2002). The “type variety” of P. destructiva var. destructiva was assigned to section Phoma whereas the other Phoma reported on tomato were allocated to other sections: Phoma lycopersici to section Boeremia and Phoma radicina to section Paraphoma (Chen et al. 2015; Gruyter et al. 2012). The ambiguity of some section designations has been discussed by Aveskamp et al. (2010). Phoma destructiva is an example of such an ambiguity because its two varieties are accommodated in two different sections due to the presence or absence of septate conidia (Aveskamp et al. 2010). Based on morphology, COAD 2069 fits into P. destructiva var. diversispora, however, it did not group with such a variety in the phylogenetic analysis. These observations suggest that emphasis put on spore size and septation by taxonomists to decide on the placement of fungi of this group in the past may have been misleading.

Phoma blight has been an important, although sporadic problem in some countries such as the United States and India (Jones et al. 2014). Recently, Phoma was recorded causing blight on greenhouse-grown tomato in Malaysia (Rashid et al. 2016). Seed transmission of Phoma diseases is known to be important in paprika and tomato. Colonized seed and transplant seedlings are thought to be involved in long distance dissemination of the pathogen (Jones et al. 2014). It is likely that, in the case of the outbreak observed in Viçosa, infected seeds have served as the source of inoculum. Therefore, considering the high level of disease severity observed on the various tomato varieties evaluated in this study, it is important to investigate what was the original source of P. destructiva and whether it can be disseminated by infected seeds. It is possible that the spread of the pathogen onto plantation areas, particularly under greenhouse conditions, may result in serious losses, posing yet an additional threat for this highly valuable crop in Brazil, for which disease management is already a constant challenge. Additionally, a more detailed comparison between isolates of P. destructiva associated with blight and those associated with post-harvest fruit disease should be conducted in order to clarify whether they represent distinct taxa.