Vasić et al., 2019, Biologica Nyssana 10(1) 10 (1) September 2019: 17-21 DOI: 10.5281/zenodo.3463990 Phytopathogenic fungi causers fungal diseases of the faba bean (Vicia faba L.) in Serbia Original Article Tanja Vasić Faculty of Agriculture, University of Niš, Kosančićeva 4, 37000 Kruševac, Serbia tanjavasic82@gmail.com (corresponding author) Sanja Živković Faculty of Agriculture, University of Niš, Kosančićeva 4, 37000 Kruševac, Serbia gajicsanja43@gmail.com Jordan Marković Institute for Forage Crops, 37251 Kruševac, Serbia jordan.markovic@ikbks.com Ivana Stanojević Faculty of Agriculture, University of Niš, Kosančićeva 4, 37000 Kruševac, Serbia stanojevic.ivana85@gmail.com Sonja Filipović Faculty of Agriculture, University of Niš, Kosančićeva 4, 37000 Kruševac, Serbia sonjafilipovic86@yahoo.com Dragan Terzić Institute for Forage Crops, 37251 Kruševac, Serbia dragan.terzic.agro@gmail.com Received: June 24, 2019 Revised: September 8, 2019 Accepted: September 9, 2019 Abstract: Faba bean (Vicia faba L.) is one of the oldest legume crops mainly grown as a valuable protein-rich food, both for human and animal consumption, where, in latter case, it provides an alternative to soybean meals in the tem- perate regions. There has not been systematic research of faba bean mycoflora in Serbia. This paper aims to present the results of preliminary investiga- tion of mycopopulation of 10 different genotypes of faba bean. Total of 400 plant parts were examined, and 6 genera of fungi were isolated: Fusarium, Rhizoctonia, Phoma, Sclerotinia, Alternaria and Ascochyta. The results in- dicate that faba bean is vulnerable to a large number of phytopathogenic fungi that can have a significant impact on reducing its yield and quality. Key words: faba bean, mycopopulation Apstract: Fitopatogene gljive izazivači gljivičnih oboljenja boba (Vicia faba L.) u Srbiji Bob (Vicia faba L.) je najstarija kultura koja se uzgaja kao hrana bogata proteinima, kako za ljudsku, tako i za stočnu ishranu, predstavljajući al- ternativu sojinim obrocima u umerenim područjima. S obzirom da nije bilo sistematskog istraživanja mikoflore boba u Srbiji, cilj ovog rada je da se predstave preliminarni rezultati mikopopulacije 10 različitih genotipova boba. Ispitano je ukupno 400 biljnih delova i izolovano je 6 rodova gljiva: Fusarium, Rhizoctonia, Phoma, Sclerotinia, Alternaria i Ascochyta. Do- bijeni rezultati ukazuju da je bob osetljiv na veliki broj fitopatogenih glji- va koje mogu imati značajan uticaj na smanjenje prinosa i kvaliteta boba. Ključne reči: bob, mikopopulacija Introduction Faba bean is an annual plant from the genus of vetches (Vicia), the family of legumes (Fabaceae). It is originally of African-Asian origin, and it is grown all over the world. Faba bean was known as an an- nual field legume since ancient times. Faba bean was cultivated in ancient Egypt, Greece, and Rome, as confirmed by numerous records. It was used for hu- man nutrition as well as for green fertilisation. To- day, faba beans, both garden and fodder, are grown in many countries of the world. In Europe it is cul- tivated in England, Belgium, Scandinavian coun- tries, Germany, Russia and Italy (Mišković, 1986). In our country faba bean is grown in a low capacity, especially as a fodder. Faba bean is a versatile fod- der crop. The content of nutrients in the grain of the faba bean and the plant as a whole is significant. The grain contains about 85% dry matter, 25.1% of crude proteins, 46.8% of BEM, 1.6% of crude fat, 9.4% of raw cellulose and about 3.5% of crude ash. Because of this composition, faba bean serves as an excellent concentrate feed for most species of livestock. Also, fabae bean grain is used in human nutrition, like stew, as it has a high nutritional value (Vučković, 1999; Aleksić et al., 2015). Vicia faba L. can fix ni- trogen through symbiosis with Rhizobium legumi- nosarum in its root nodules (Stoddard et al., 2010). The most important and widespread fungal dis- eases observed at all locations in the world are: rust (Uromyces fabae), chocolate spot (Botrytis fabae and B. cinerea), ascochyta blight (Ascochyta fabae), leaf © 2019 Vasić et al. This is an open-access article distributed under the terms of the Creative Commons At- tribution License, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. 17 spots (Alternaria alternata and Cercospora fabae) and wilt/root rot complex (Fusarium oxysporum and Macrophomina phaseolina dominating). Other dis- eases of apparent minor importance recorded at low incidence levels at most locations in the world are Sclerotinia blight (Sclerotinia sclerotiorum), downy mildew (Peronospora viciae) (Akem and Bellar, 1999; Sillero et al., 2010; Stoddard et al., 2010). In Ethiopia chocolate spot (Botrytis fabae and B. cin- erea) and rust (Uromyces fabae) are the major dis- eases which can reduce yield by about 61 and 21%, respectively (Tegen, 2017). In Croatia two Fusari- um species were determined on the seed vetch, F. verticillioides and F. proliferatum (Miličević et al., 2013). Since faba bean has become very important in recent years as a fodder crop in Serbia, the aim of this paper was to determine the phytopathogenic fungi disease causative agents in faba bean for more precise understanding of the problems (plant die-off, reduction of yield and quality, etc.) that arise as a result of the presence of phytopathogenic fungi in faba bean. Materials and methods The samples were collected between March and June 2016-2017 at the location of the Institute for forage crops in Globoder. The samples of faba bean seed for the study of mycoflora were taken from differ- ent sites: the Rasina region (Kruševac 1, Kruševac 2, Gaglovo 2), Zaječar region (Crni kao), Nišava re- gion (Praskovče, Rujevica, Šarbanovac, Sokobanja) and Pomoravlje region (Jagodina 3, Oparić). Parts of plants are carefully washed under running water. Af- ter washing, the parts of stem and roots were cut to pieces of 0.5-1 cm in size. Prepared samples of roots and stems were disinfected with 96% ethanol for 10 seconds and with 1% sodium hypochlorite (NaOCl) for 1 minute and then washed three times in sterile distilled water. They were then dried on sterile filter paper and placed on potato dextrose agar (PDA) with streptomycin. Five pieces of the plant parts (root and stem) were each placed in Petri dish in four replica- tions. They were kept in a thermostat at 25°C in 12 h light / 12 h dark regime. The observations were per- formed every 3 days, and the majority of mycelium samples were developed up to 14 days. Developed mycelia were screened to a new PDA substrate and, after an initial grow, the peak part of the mycelium was reseeded on PDA again. Microscopic exami- nation was performed using microscopes Olympus CX31. Morphological identification of fungi to the genus was carried out using a standard key. The fre- quency of isolation was calculated in percents ac- cording to the formula by Vrandečić et al. (2011): Results and discussion In these studies, the mycopopulation of faba bean genotypes was examined in a total of 400 plant parts. The isolation frequency of the pathogens from dis- eased samples largely confirmed the field diagno- sis based on visual symptoms. There were clearly expressed symptoms on the leaves in the form of necrotic spots and lesions in all the plants where fungi were isolated. Fungi of genera Ascochyta and Alternaria were isolated from these plants (Tab. 1). Likewise, necrosis with the presence of white, aerial mycelium was observed on stems in a large number of plants. Fungi from the genus Sclerotinia were isolated from the lower third of the stems of these (Tab. 1). Symptoms in the form of light to dark brown necrosis were present at the plant root system, and from these plants were isolated fungi from the gen- era Fusarium, Phoma and Rhizoctonia (Tab. 1). In these studies, there was a difference in the iso- lation frequency of some genera of phytopathogenic fungi in faba bean genotypes originating from differ- ent regions of Serbia. In the samples from the Rasina region (Kruse- vac 1, Krusevac 2, Gaglovo 2), on the leaves, the infection by genus Alternaria was 10-40%, and by genus Ascochyta was represented in all tested sam- ples at 30% (Tab. 1). Fungi from genera Fusarium with 66.67% and Phoma with 40% were dominant in the crowns of the root in the faba bean plants from Rasina region (Tab. 1). In the Zaječar region (Crni Kao), the most represented fungi were Ascochyta with 40% in leaves, and Sclerotinia with 43.33% it roots (Tab. 1). In the Nišava region (Praskovče, Rujevica, Šarbanovac, Sokobanja), the prevalent species were from the genus Alternaria with 80% in leaves, while in the root system, fungi from genus Rhizoctonia were the most abundant with 46.67%. Genera Alternaria with 80% and Sclerotinia with 60% were the most prevalent in Pomoravlje region (Jagodina 3, Oparić) (Tab. 1). The genera Fusarium, Phythophthora, Rhizocto- nia, Phoma, Verticillium, Alternaria and Sclerotinia were dominant in annual and perennial legumes in the world (Tivoli et al., 2006; Villegas-Fernández and Rubiales, 2011; Salam et. al., 2011; Sillero et al., 2014; O’Sullivan and Angra, 2016). Miličević et al. (2013) determined two Fusarium species, F. verticil- lioides and F. proliferatum in vetch seed in Croatia. Rhizoctonia solani Kühn is a soil parasite that can 18 BIOLOGICA NYSSANA ● 10 (1) September 2019: 17-21 Vasić et al. ● Phytopathogenic fungi causers fungal diseases of the faba bean (Vicia faba L.) in Serbia (%) Isolation frequency Number of segments containing the fungal species Total number of segments used in the isolation x 100= cause serious problems on many legumes, especial- ly on faba bean (Assunção, 2011). In Canada, 304 faba bean genotypes were tested for resistance to R. solani and only five were identified with high resist- ance (Rashid and Bernier, 1993). Sclerotinia stem rot, a fungal disease caused by Sclerotinia trifoli- orum, is often a serious problem in faba beans (Vicia faba) in Greece (Lithourgidis et al., 2005). Chocolate spot is caused by Botrytis cinerea Pers. and B. fabae Sard., the latter being the most important since its action can result in serious plant damage, which is not usually the case with B. cin- erea. Chocolate spot is especially severe in humid areas, having been reported to be the cause of heavy reductions in yields in places such as the Maghreb, Southern China, Egypt, UK or France (Tivoli et al., 2006). The type of Alternaria tenuissima was detected on the broad bean in Japan. The disease was found in all surveyed fields. The initial lesion was brown, water-soaked, circular to slightly irregular. Then the lesion enlarged and became concentric. Mature 19 leaves had coalescing necrosis surrounded by yel- lowing. Older leaves of the plant were particularly affected. In a later stage of the disease, the leaves became blighted from the margin to the centre and most of the diseased plants defoliated (Rahman et al., 2002). Ascochyta blight, caused by Ascochyta fabae Speg., is a common and destructive disease of faba bean (Vicia faba L.) in the Middle East, Eu- rope, Canada, New Zealand (Díaz-Ruiz et al., 2009). In Syria Ascochyta blight were frequently isolated from infected samples showing typical chocolate spot symptoms (Akem and Bellar, 1999). Symptoms occur on leaves, stems and pods of infected plants, and can be confused with the early stages of choco- late spot (Botrytis fabae). On leaves, small, circular, dark-brown spots appear first. As the disease devel- ops, lesions enlarge and turn light and then change to dark grey. They become irregular in shape, often zonate, and may coalesce to cover most of the leaf surface. Leaf tissue next to the lesions may become black and necrotic. Within the lesions, numerous pinhead-sized black fruiting bodies (pycnidia) of Genotypes Number of samples (Plant part) Fungi species (leaf) (%) Isolation frequency Fungi species (root) (%) Isolation frequencyLeaf Root 3/II Jagodina 3 10 30 Alternaria sp. 80 Sclerotinia sp. 60 19 red Rujevica- Sokobanja 10 30 Alternaria sp. 80 Sclerotinia sp. Rhizoctonia sp. 26.67 20 41 red Kruševac 1 10 30 Alternaria sp. Ascochyta sp. 30 30 Fusarium sp. 66.67 7/II Oparić 10 30 Alternaria sp. Ascochyta sp. 30 10 Fusarium sp. Sclerotinia sp. 23.33 6.67 15 red Šarbanovac- Sokobanja 10 30 Alternaria sp. Ascochyta sp. 60 10 Fusarium sp. Sclerotinia sp. 43.33 16.67 20 red Crni Kao 10 30 Alternaria sp. Ascochyta sp. 20 40 Sclerotinia sp. 43.33 Kruševac 2 10 30 Alternaria sp. Ascochyta sp. 10 30 Phoma sp. Rhizoctonia sp. 40 23.33 10 red Gaglovo 2 10 30 Alternaria sp. Ascochyta sp. 40 30 Rhizoctonia sp. Fusarium sp. 33.33 26.67 4 red Sokobanja 10 30 Alternaria sp. Ascochyta sp. 50 20 Rhizoctonia sp. 46.67 13 red Praskovče 10 30 Alternaria sp. Ascochyta sp. 30 30 Fusarium sp. Sclerotinia sp. Rhizoctonia sp. 16.67 13.33 13.33 Table 1. Frequency of fungal isolation on Vicia faba L. BIOLOGICA NYSSANA ● 10 (1) September 2019: 17-21 Vasić et al. ● Phytopathogenic fungi causers fungal diseases of the faba bean (Vicia faba L.) in Serbia 20 the fungus develop. These appear only under moist conditions and are often concentrically arranged (El- Komy, 2014). Ascochyta blight is a common disease that causes up to 90% yield losses in susceptible cul- tivars when environmental conditions are favourable for disease development (Díaz-Ruiz et al., 2009). Conclusion This paper presents preliminary results of mycopop- ulation of 10 experimental faba bean genotypes. The obtained results indicate that faba bean is susceptible to the attack by a large number of phytopathogenic fungi that can significantly reduce its yield and af- fect its quality. Faba bean has more and more signifi- cance in our country, becoming an important fodder crop as livestock feed. This paper is the beginning of a more comprehensive study of phytopathogenic fungi in faba bean. So far, there has been no signifi- cant research in this field in Serbia, so future inves- tigations related to the selection of genotypes with increased tolerance to the disease-causing agents will be conducted. Selection to disease resistance can be used as part of the Integral Plant Protection program (IPP), which can include biological and ag- ronomic measures, as well as cultivation of resist- ant cultivars and hybrids, and which aims to prevent economically significant damage and preserve the environment. 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