Moumni Et Al., 2019 Phytopathologia Mediterranea Black Rot of Squash (Cucurbita Moschata Duchesne) Caused by Stagonosporopsis Cu
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Moumni et al., 2019 Phytopathologia Mediterranea 1 Black rot of squash (Cucurbita moschata Duchesne) caused by Stagonosporopsis 2 cucurbitacearum reported in Italy 3 4 Marwa Moumni1,2,3, Valeria Mancini1, Mohamed Bechir Allagui3, Sergio Murolo1, and 5 Gianfranco Romanazzi1* 6 7 1 Department of Agricultural, Food and Environmental Sciences, Marche Polytechnic 8 University, Via Brecce Bianche, 60131 Ancona, Italy 9 2 National Agricultural Institute of Tunisia, 43 Avenue Charles Nicolle, 1082 Tunis, 10 Tunisia 11 3 Laboratory of Plant Protection, National Institute for Agronomic Research of Tunisia, 12 University of Carthage, Rue Hédi Karray, 2080 Ariana, Tunisia 13 14 *Corresponding author: G. Romanazzi 15 E-mail: [email protected] 16 Tel: +39-071-2204336 17 18 Running title: Black rot on squash in Italy 19 1 Moumni et al., 2019 Phytopathologia Mediterranea 1 Summary 2 3 Stagonosporopsis cucurbitacearum (syn. Didymella bryoniae) is a major pathogenic 4 fungus that can affect cucurbits through induction of the disease known as black rot. 5 This pathogen produces irregular white spots covered with pycnidia on the infected 6 fruit. Twenty squash fruit (cv. ‘Butternut’) with symptoms that can be ascribed to S. 7 cucurbitacearum were collected in Italy from two locations: Osimo (AN) and 8 Montacuto (AN), in the Marche region. Several colonies were isolated from these fruit, 9 the morphological features of which corresponded to S. cucurbitacearum. 10 Morphological identification was confirmed by molecular tools (multiplexing of three 11 microsatellite markers) and by sequence analysis using internal transcribed spacers. The 12 sequence identity for the internal transcribed spacer regions was over 98% compared 13 with sequences of S. cucurbitacearum in the NCBI database. To our knowledge, this is 14 the first report of S. cucurbitacearum on Cucurbita moschata fruit with black rot 15 symptoms in Italy. 16 17 Key words: Butternut squash; first report; black rot; ITS sequencing; microsatellite 18 markers 19 2 Moumni et al., 2019 Phytopathologia Mediterranea 1 Introduction 2 3 Black rot (BR) is caused by the fungal pathogen Stagonosporopsis cucurbitacearum 4 (Fr.) Aveskamp, Gruyter & Verkley (Aveskamp et al., 2010) (anamorph Phoma 5 cucurbitacearum (Fr.) Sacc.) synonym Didymella bryoniae (Fuckel) Rehm, which is 6 one of the most devastating pathogens on cucurbits worldwide (Li et al., 2015; Yao et 7 al., 2016). S. cucurbitacearum and S. citrulli can infect several species of the 8 Cucurbitaceae family, including watermelon (Citrullus lanatus (Thunb.) Matsum. & 9 Nakai) (Rennberger and Keinath, 2018; Babu et al., 2015; Huang and Lai, 2019), 10 muskmelon (Cucumis melo L.) (Nuangmek et al., 2018), squash (Cucurbita maxima L., 11 Cucurbita moschata Duch) (Keinath, 2014), and pumpkin (Cucurbita pepo L.) (Grube 12 et al., 2011). They can cause infection of the stems, leaves, roots, seeds, and fruit 13 (Keinath, 2011). 14 Infected fruit manifest large irregular-shaped spots and black rot (Choi et al., 15 2010). Fruiting bodies are found in the oldest parts of lesions, because S. 16 cucurbitacearum is a necrotrophic fungus (Keinath, 2014). Keinath (2011) reported that 17 S. cucurbitacearum produces black mycelia inside melon and giant pumpkin (C. 18 maxima) fruit. The ideal conditions for disease development include humidity over 19 90%, leaf wetness and temperatures from 16 °C to 24 °C (Park et al., 2006; Seebold, 20 2011). BR can reduce preharvest and postharvest yields (de Neergaard, 1989), and 21 cause up to 15% fruit loss (Keinath, 2000). S. cucurbitacearum has only been reported 22 once in Italy, in 1885 on C. melo L., and it was described as Didymella melonis Pass. by 23 Giovanni Passerini (Corlett, 1981). Our investigations were aimed at the identification 24 of the causal agent of black rot symptoms on squash fruit. 25 3 Moumni et al., 2019 Phytopathologia Mediterranea 1 Materials and methods 2 Fungal isolation 3 Twenty fruit of squash (C. moschata; cv. ‘Butternut’) with symptoms of black rot were 4 collected from two locations in September and October 2018: Osimo (AN) and 5 Montacuto (AN), in the Marche region (Italy). Small infected pieces of squash peel (~2 6 mm) were cut from the fruit, sterilized with 1% sodium hypochlorite for 2 min, washed 7 three times with sterilized distilled water, placed into 90-mm Petri dishes containing 8 water agar (Bacteriological agar; Liofilchem, Italy), and incubated at 24 °C. After 7 9 days, the pycnidia were excised, placed into Petri dishes containing potato dextrose agar 10 (Liofilchem, Italy), and incubated at 24 °C. Morphological identification was carried 11 out according to the color and shape of the colonies, and to the size of the spores (50 12 spores measured). 13 14 DNA amplification and phylogenetic studies. 15 The fungal genomic DNA was extracted from 100 mg mycelia of isolates grown on 16 potato dextrose agar as pure cultures, following the protocol proposed by Varanda et al. 17 (2016). The DNA was amplified in a rapid PCR based assay for distinguishing the three 18 morphologically similar species (S. cucurbitacearum, S. citrulli, and S. caricae) by 19 multiplexing of three microsallite markers Db01, Db05 and Db06 (Brewer et al., 2015). 20 Then, the primer pairs ITS1 and ITS4 (White et al., 1990) were used to amplify the 21 internal transcribed spacers (ITS). The PCR products were separated on 1.5% agarose 22 electrophoresis gels, stained with Red Gel (Biotium, Hayward, CA, USA), and 23 visualized, with images captured using an imagining system (Gel Doc XR; BioRad). 24 Selected PCR amplicons were purified and sequenced by Genewiz UK, and have been 25 deposited with Genebank (accession numbers: isolates ID1, MK330934; ID3, 4 Moumni et al., 2019 Phytopathologia Mediterranea 1 MK330935; ID9, MK330936; for ITS regions). Finally, the nucleotide sequences were 2 subjected to Blast analysis to determine the relative identities with other sequences 3 available in Genebank. 4 5 Results and discussion 6 7 In the two surveyed locations in Italy, black rot symptoms occurred on the butternut 8 squash fruit. The initial symptoms were brown circular spots with exudates on the 9 squash peel (Figure 1A). Over time, the spots became white and were covered with 10 black fruiting bodies (Figure 1B, C). After 8 days of incubation on water agar, under the 11 stereomicroscope, some pycnidia were seen to be developing in rows on the fruit peel 12 (Figure 2A). On crushing the pycnidia, the pycnidiospores inside were seen to be 13 cylindrical, mostly nonseptate, with a few 1-septate, as 4 µm to 11 µm × 2 µm to 5 µm 14 in size (Figure 2B, C). The isolates on potato dextrose agar showed white mycelia on 15 the top and black mycelia on the underneath. These morphological characteristics 16 coincided with those known for S. cucurbitacearum (Keinath et al., 1995; Koike, 1997; 17 Choi et al., 2010; Keinath, 2013). The morphological identification was then supported 18 by the multiplex amplification using the primers Db01f/r, Db05 f/r and Db06 f/r, 19 which yielded two amplicons (220 bp and 280 bp), characteristic for S. 20 cucurbitacearum. The presence of an amplicon of 280 bp and the lack of an amplicon of 21 about 360 bp excluded that the isolates ID1, ID3, and ID9 were S. caricae or S. citrulli, 22 as reported by Brewer et al. (2015) (Figure 3). Moreover, the Blast analysis showed 23 high sequence identity (98%-99%) for the ITS regions compared to other sequences of 24 S. cucurbitacearum already in the NCBI database, as reported in Table 1. Therefore, the 5 Moumni et al., 2019 Phytopathologia Mediterranea 1 isolates ID1, ID3, and ID9 from butternut squash are confirmed as Stagonosporopsis 2 cucurbitacearum. 3 Stagonosporopsis spp. is a major pathogen of cucurbits worldwide, and it occurs 4 everywhere they are grown (Nuangmek et al., 2018; Stewart et al., 2015; Mancini et al. 5 2016). Gummy stem blight and BR can affect all part of cucurbits, including stems, 6 leaves, roots, seeds, and fruit. Furthermore, this pathogen is seedborne and soilborne, 7 and it can remain for long periods in the seeds and in the soil, thus continuing to be 8 spread around the world through infected seed (Keinath 2011). Seedborne pathogens 9 can reduce of the quantity and quality harvested fruits and/or seeds, and their 10 management is crucial for a profitable production (Mancini et al., 2014). On cantaloupe, 11 field losses due to S. cucurbitacearum can reach 100% under conditions conducive to 12 infection (Nuangmek et al., 2018), and on watermelon, Gummy stem blight and BR can 13 cause significant production losses both in the field and post-harvest (Maynard and 14 Hopkins, 1999). No commercial cultivar of any cucurbit species that has resistance to 15 Gummy stem blight is available on the market (Keinath, 2017). 16 Somai et al. (2002) have already reported S. cucurbitacearum for butternut 17 squash in the USA. In Italy, this pathogen has been reported on C. melo (Corlett, 1981). 18 To our knowledge, this is the first report of Stagonosporopsis cucurbitacearum on 19 squash in Italy. 20 21 6 Moumni et al., 2019 Phytopathologia Mediterranea 1 Literature cited 2 3 Aveskamp M. M., J. de Gruyter, J. H. C. Woudenberg, G. J. M. Verkley and P. W. 4 Crous, 2010. Highlights of the Didymellaceae: a polyphasic approach to 5 characterize Phoma and related pleosporalean genera. Studies in Mycology 65, 6 1−60. 7 Babu B., Y. W. Kefialew, P. F. Li, X. P. Yang, S. George, E. Newberry, N. Dufault, D. 8 Abate, A. Ayalew, J. Marois and M. L. Paret, 2015. Genetic characterization of 9 Didymella bryoniae isolates infecting watermelon and other cucurbits in Florida 10 and Georgia. Plant Disease 99, 1488−1499. 11 Brewer M. T., M. Rath, H. X. Li, 2015.