Accepted Manuscript

Accepted Manuscript This is a PDF file of an unedited manuscript that has been accepted for publication in the journal Ratarstvo i povrtarstvo. This early version of the accepted manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the editing production process errors may be discovered which could affect the content. Manuscript submitted: 15 July 2020 Manuscript accepted: 19 August 2020 doi: 10.5937/ratpov57-27516 To appear in: Ratar. Povrt. (2020) ------------------------------------------------------------------------------------------------------------- original scientific paper Diversity of phytopathogenic fungi on soybean seed in Serbia Slobodan Krsmanović* · Kristina Petrović · Marina Ćeran · Vuk Đorđević · Predrag Ranđelović · Simona Jaćimović · Zlatica Miladinov Institute of Field and Vegetable Crops, 30 Maksima Gorkog, 21000 Novi Sad, Serbia Corresponding author: [email protected] Current title: Krsmanović S. et al. Diversity of phytopathogenic fungi on soybean seed in Serbia Acknowledgments: The research was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, grant number: 451-03-68/2020-14/200032. Summary: Soybean is one of the most important industrial crops in the world. The rich nutritional content of the seed is the reason for the increased cultivation of this crop all over the world. However, a large number of phytopathogenic fungi that exist in soybean seed can reduce the nutritional content, germination, and seed vigor. Health status analysis of soybean seed has shown that Peronospora manshurica, Macrophomina phaseolina, Botrytis cinerea, Cercospora kikuchii as well as the species from genera Alternaria, Diaporthe, and Fusarium colonized soybean seed in Serbia over three years (2016-2018). Species from the genus Alternaria and Peronospora manshurica were dominant in all three examined years. Furthermore, it has been noticed that weather conditions, location, and cultivar significantly influenced the intensity of the infection. The results of this study have shown which pathogens pose a threat to successful soybean seed production and help in finding preventive measures to control these pathogens during vegetation. Keywords: soybean, seed, phytopathogenic fungi, incidence of infection Introduction Soybean (Glycine max (L.) Merr.) is the most commonly cultivated protein-oil crop in the world, and in Serbia as well, primarily because of the seed which is one of the major food and feed sources (Vlahović et al., 2013). Thus, the production of high-quality soybean seed is very important, which can be improved by right agronomic measures such as sowing a certified, disease-free seed. To keep the high health status of the seed, it is necessary to apply crop rotation which helps to break the disease cycle in the field and thus increase yield. Crop rotation of soybean and crops that share common diseases such as sunflower, as well as cultivation in monoculture, result in increasing inoculum source and the seed infection frequency by pathogens (Vidić et al. 2006). More than 200 phytopathogenic microorganisms (primarily fungi) have been detected on soybean (Hartman et al. 2015), and it is considered that about 30 species can cause significant economic damage (Roy et al. 2000). Most of them colonize soybean seed and thus spread and distribute in other soybean-growing regions worldwide (Backman et al., 1985; Pioli et al., 1999; Vidić et al., 2006). The most common fungi of soybean seed worldwide are species from following genera: Diaporthe, Fusarium, and Alternaria as well as species: Cercospora kikuchii, Rhizoctonia solani Kühn, Sclerotinia sclerotiorum, Botrytis cinerea Persoon, Macrophomina phaseolina, and Peronospora manshurica (Wrather et al., 2010; Tenuta et al. 2015). Phytopathogenic fungi, such as species of genus Diaporthe, negatively affect germination and seed quality (Petrović et al. 2015; 2016; 2018). Seed infections can be with no visible symptoms (latent infection) or with typical symptoms such as rot, mycelia, or discoloration. In the U.S. during the period from 2006 to 2009, seedling diseases were on the second place of importance as a causative factor in reducing the soybean yield (Koenning & Wrather, 2010). In top eight soybean-producing countries during 2006, soybean yield was mostly affected by Septoria glycines Hemmi, Cercospora kikuchii (Matsumoto & Tomoyasu) Gardner, Peronospora manshurica (Naumov) Sydow, Macrophomina phaseolina (Tassi) Goidànich, Sclerotinia sclerotiorum (Libert) de Bary, Phakopsora pachyrhizi Sydow & Sydow and with phytopathogenic fungi from the genus Diaporthe and Fusarium species complex (Wrather et al., 2010). In Serbia, the most prevalent pathogen in soybean seed in the past was P. manshurica, followed by species from following genera: Alternaria, Fusarium and Diaporthe (Medić-Pap et al., 2007; Petrović & Vidić, 2010; Lević et al., 2012). High humidity at the time of maturation and soybean harvest can additionally increase the harmfulness or seed infection degree with phytopathogenic fungi. Soybean yield losses caused by diseases varied due to weather and climatic conditions in most countries. The dry areas are favorable for diseases such as charcoal rot caused by M. phaseolina (Wrather & Koenning, 2006). Wet weather is suitable for developing Sclerotinia stem rot in the U.S. and Canada, Rhizoctonia blight in India, and anthracnose in the central and northern portions of Brazil and Paraguay (Wrather et al., 2010). Besides, wet weather at harvest time enhanced Diaporthe seed decay in Canada (Xue et al., 2007). Since soybean production in Serbia and soybean seed trade in Europe has rapidly increased in the last several years, the spread of the diseases by seed can be expected. The aim of this study is to make a profile of the phytopathogenic fungi that colonizes soybean seed in the main regions of soybean production in Serbia. Materials and Methods Isolation and identification of seed phytopathogenic fungi Soybean seed samples were collected from experimental fields in seven locations in Vojvodina Province, Serbia in the period 2016-2018. Three cultivars from different maturity groups (MG) were examined: ‘Galina’ (MG=0), ‘Sava’ (MG=I), and ‘Rubin’ (MG=II). First, samples of 100 seeds per cultivar were previously studied for the presence of an obligate host-specific pathogen P. manshurica using binocular loupe. Afterward, seeds were surface-disinfested in 4% sodium hypochlorite for 5 min, washed two times in sterile water, and dried on a filter paper. All 100 seeds were placed in 20 Petri dishes with standard potato dextrose agar medium (PDA), five seeds in each, and incubated at 22 ± 2°C for seven days, under 12 h of alternating light/dark conditions. Fungi colonies were developed from infected seed and transferred on fresh PDA medium and incubated in a thermostat at 23 ± 2°C. The fungal pathogens of the soybean seed were profiled using morphological and cultural features. The first assessment was performed on the fourth day after isolation by observation of macro- and microscopic morphological features of mycelium and sporulation structures that were formed. Some of the colonies were left more days for incubation until identified. Pathogen identification performed using naked-eye detection of cultural and macro morphological characteristics of phytopathogenic fungi, such as colony appearance, the color of mycelium, and asexual and sexual structures, if the fungi have formed them. Afterwards, the morphological features such as the type of conidia, their length and shape were studied in detail under a light microscope at different magnifications (x10, x20, and x40). As a basis for comparing the results of the identification, it was used the Compendium of Soybean Diseases and Pests, Fifth edition (Hartman et al., 2015), where all the listed morphological features of genera and their species are precisely described that were found in this study. The incidence of fungal pathogens per sample of soybean seeds is presented in percentage, and the results were calculated with the following formula: 100 푁푢푚푏푒푟 표푓푡ℎ푒 푛푓푒푐푡푒푑 푠푒푒푑푠 푏푦 푓푢푛푔푎푙 푠푝푒푐푒푠 표푟 푔푒푛푢푠 푥 푇표푡푎푙 푛푢푚푏푒푟 표푓 푠푒푒푑푠 푛 푎 푠푎푚푝푙푒 Results and discussion Data obtained from three consecutive years have shown differences in the incidence of phytopathogenic fungi on the soybean seeds compared to a year, location, and cultivars (Fig. 1, 2, and 3). Figure 1. The incidence of fungal pathogens on the soybean seed of cv. ‘Galina’ (%) Figure 2. The incidence of fungal pathogens on the soybean seed of cv. ‘Sava’ (%) Figure 3. The incidence of fungal pathogens on the soybean seed of cv. ‘Rubin’ (%) The lowest fungal incidence of seed was noticed in 2017. The reason might be the absence of precipitation at the time of pod and seed development as well as plant maturity stage, which does not favor the development of the phytopathogenic population on seed (Petrović & Vidić, 2010). During 2017, the highest amount of precipitation was observed in Šabac location (Fig 4) where also, the highest seed infection was recorded on cvs. ‘Sava’ (26%) and ‘Galina’ (9%) followed by 1% of seed infection of cvs. ‘Galina’ and ‘Rubin’ in location Karavukovo. Figure 4. The sum of precipitation in the period of soybean maturation, July-September (2016- 2018) In 2016 and 2018, climatic conditions were favorable for pathogens development, and the presence of phytopathogenic fungi on soybean seeds was confirmed

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