The Spectrum of Fungal Pathogens of Sorghum Bicolor X Sorghum Sudanense

6–71RYHPEHU 2019, Brno, Czech Republic

The spectrum of fungal pathogens of Sorghum bicolor x Sorghum sudanense

Eliska Novakova, Ivana Safrankova Department of Crop Science, Breeding and Plant Medicine Mendel University in Brno Zemedelska 1, 613 00 Brno CZECH REPUBLIC [email protected]

Abstract: Sorghum belongs to the most cultivated cereals in the world. The biggest producers for food industry are Africa and Asia. In Europe it is mostly used as animal´s feed. The sorghum is a minority crop in the Czech Republic, it is cultivated mainly for silage as a forage crop for livestock production systems or for biogas production. Five evaluations were performed in 2019 (June–July) under the field condition on sorghum variety ´KWS Tarzan´. The occurrence of fungal pathogens on sorghum were observed and evaluated in the field experimental station in Žabčice. Leaves which were affected by fungal pathogens were photographed and collected for their determination. The fungal pathogens were identified according to morphological and microscopic characteristics which appeared on a leaves surface (spots, mycelium, and spores) on the field or after laboratory ´wet-cell´ cultivation. The sorghum plants were infected by pathogens from the group of leaves and stalks spots (Colletotrichum sublineola, Cercospora sorghi, Exserohilum turcicum, Bipolaris cookei) and sorghum rust (Puccinia sorghi). Key Words: Colletotrichum sublineola, Cercospora sorghi, Exserohilum turcicum, field monitoring, Sudan grass

INTRODUCTION Due to the climatic changes crops with specific attributes have to be included into the rotation in the Czech Republic. Such crops have to be able to provide good and high crop yields at high temperatures during the summer months and droughts. The crop which meets the requirements is sorghum (Sorghum bicolor × Sorghum sudanense). It is also known as Sudan grass and is suitable for feed and biogas stations. The limiting factor for its cultivation is low temperatures during sowing and early emergence (Adamčík and Tomášek 2016). This problem can manifest as low coverage growth, high degree of weed infestation or lower resistance to harmful organisms. These factors can have negative influence on the quantity and quality of production (Das 2018). Fungal pathogens of sorghum can be sorted into three groups according to parts of plants which are infected. There is an occurrence of inflorescence and seed pathogens (smuts and Fusarium spp.), foliar and stalk pathogens (leaves spot and rusts), and the root and root disc pathogens (Macrophomina phaseolina). Every pathogen which is mentioned above should make a lower biological value of feed → lower photosynthetic activities, lower digestibility for cattle as a consequence of lower content of saccharides and mycotoxin production (Nedělník et al. 2011). The most important sorghum pathogens in Europe are Colletotrichum sublineola, (Baroncelli et al. 2014, Das 2018), Ascochyta sorghi (Schuh et al. 1986), Bipolaris cookei (Jonar et al. 2011, Das 2018), Cercospora sorghi, Ramulispora sorghicola, Exserohilum turcicum (Kuthan 2010), so the aim of this study was to determine, if they are present in the Czech Republic, nowadays.

MATERIAL AND METHODS The small-plot experiment was established at the field trial station in Žabčice 49°00'57.614" N, 16°5'92.1042 E" in Southern Moravia (Figure 1a–b). The average annual temperature is 9.5 °C, and average annual precipitation is 480 mm. The course of weather in the year 2019 is given

87 6–71RYHPEHU 2019, Brno, Czech Republic in Figure 2. The soil of experiment field is sandy. One plot area was 30 m2. Sorghum seeds were sown on 25. 4. 2019 (25 germinative seeds/m2), without pre-crop. The effect of nitrogen and sulphur fertilization was observed. The fertilizer with nitrogen (N) and sulphur (S) applied 7 days after plant emergence (27 May 2019), zinc foliar application (Zinkosol Forte) was made in 8–leaf growth stage (26 June 2019). The fertilizer with nitrogen (N) → ALZON neo-N (N = 115 kg/ha), fertilizer with nitrogen and sulphur (N+S) → ENSIN (N = 117 kg/ha, S = 58.5 kg/ha), fertilizer with nitrogen and zinc (N+Zn) → ALZON neo-N + ZINKOSOL forte (N = 115 kg/ha, Zn = 450 g/ha), fertilizer with nitrogen and sulphur and zinc → ENSIN + ZINKOSOL forte (N = 115 kg/ha, S = 58.5 kg/ha, Zn = 450 g/ha) were applied in particular variants. These plots were observed and evaluated separately from phytopathologic perspective. Figure 1 Experimental field with Sorghum bicolor × Sorghum sudanense, (https://www.google.com/maps, 2019)

Figure 2 Climate diagram, Žabčice (Meteorological station in Žabčice, Department of Agrosystems and Bioclimatology, Mendel University in Brno, 2019)

Evaluation of the occurrence of pathogens during vegetation The total health condition of growth and occurrence of sorghum pathogens was evaluated in June–July 2019 [10. 5., 20. 6., 10.7, 20. 7., and 31.7.; (Figure 3)]. Leaves which showed symptoms of the disease were photographed and collected to a paper bag and they were labelled according to a field number and fertilizer variant. These samples were used for microscopic identification of the pathogen according to morphological characteristic. The small-plot experiment was harvested by hands 1. 8. 2019.

88 6–71RYHPEHU 2019, Brno, Czech Republic

Figure 3 Growth of Sorghum bicolor × Sorghum sudanense ´KWS Tarzan´

Legend: 3a field of sorghum – Žabčice 20. 6. 2019; 3b fielf of sorghum – Žabčice 10. 7. 2019 Determination of sorghum pathogens in the lab conditions The infected leaves were cut to pieces and the surface was disinfected with 0.2% sodium hypochloride followed by 3 minutes of soaking. Then the leaves were washed by distilled water for 3 minutes, dried in sterile conditions (flow-box, sterile filtration papers, 5 minutes) and placed on Petri dish (Ø 15 cm) with two-tier filtrate paper which was moisten by distilled water. The cultivation was conducted under standard laboratory conditions (temperature 20/25 °C, light mode 14/10 hour) for 7 days. Pathogens were identified under microscope (Olympus BX12, Olympus BX41) by their morphological characteristics (size of spots on the leaves, size of spores etc.). If it was not possible to identify the pathogen directly from the leaves, cultivation of the pathogen had to be done in cultured dirt of Potato Dextrose Agar (PDA), Sabouraud agar (SA), Malt extract agar (MEA), cultivation lasted for 7–10 days, (23/20 °C, 14/10 hours light mode)

RESULTS AND DISCUSSION Monitoring and detection of sorghum pathogens in field conditions The vegetation beginning was very slow, because in May the weather was cold and wet (Figure 2), this not was suitable for sorghum growth. The sorghum stand was sparse and weedy. The first occurrence of fungal pathogens Colletotrichum sublineolum (Figure 4 a–b) and Cercospora sorghi on sorghum plants was noticed in the first half of July (Table 1). In the next observation (the second half of July) another three pathogens were determinated: Exserohilum turcicum, Bipolaris cookei (Figure 4g–h) and Puccinia sorghi (Figure 4e–f). Any differences in rate of infected plants between particular small-plot plots were not observed. Anthracnose caused by Colletotrichum sublineolum was disease with the highest occurrence on the sorghum plants in the small-plot experiment. Typical anthracnose symptoms are circular- elliptical dark spots 2–6 cm diam., with a red pigmentation on leaves and stalks (Figure 4 a–b). The centre of mature lesions is straw-colored and contains numerous acervuli with black seta (Figure 4c–d). According to Thakur et al. (2007) grey/cream/salmon-colored spore masses are produced under humid conditions. In many instances leaves can be entirely blighted, and when the blight attacks the stalks it is known as 'stalk rot'. The optimal conditions for anthracnose occurrence are when dry and moisture period take turns. Another fungal pathogen Cercospora sorghi causes very similar symptoms. The lesions are mostly isolated but can grow continuously to give long stripes. They are dark purple with a brown centre. Leaf spots which enlarge to become rectangular lesions (Odvody 1986, Cuevas 2016 et al.).

89 6–71RYHPEHU 2019, Brno, Czech Republic

Under our field conditions the same spots as Cuevas et al. (2016) described were observed → long stripes on the leaves blades, stromata were observed in a lab condition by microscopic methods. The pathogens determination was carried out on the grounds of Crous et al. (2006) study. Figure 4 Fungal pathogens of Sorghum bicolor × Sorghum sudanense

Legend: 4a–d Colletotrichum sublineolum on infected stalk (a) and infected leaf (b), setae emerging from acervuli (c–d); Puccinia sorghi on infected leaf (e) and urediniospore (f); 4g–h Bipolaris cookei conidia Bipolaris cookei causes similar symptoms as Cercospora sorghi, but the lesions are redder, but in a field conditions it is not possible to determine differences. The typical brown elliptical conidies (Figure 4g–h) were observed in lab conditions. These same symptoms and microscopic structures were described by Manamgoda et al. (2014). The last mentioned pathogen, Exserohilum turcicum has small spots that enlarge and coalesce resulting in the wilting of the young leaves. On adult plants long, elliptical, reddish purple or yellowish lesions develop, first on lower leaves and later progresses to upper leaves and stem as well. In humid weather numerous greyish black spores produced in the lesions in concentric zones. This description is with conformity with Thakur´s et al. (2007). Table 1 The particular occurrence of sorghum pathogens during a monitoring in 2019

date 10. 5. 20. 6. 10. 7. 20. 7. 31. 7. 2019 2019 2019 2019 2019 pathogen

Colletotrichum sublineola - - + + + Cercospora sorghi - - + + + Exserohilum turcicum - - - + + Puccinia sorghi - - - + + Bipolaris cookei - - - + + Legend: - negative occurrence; + positive occurrence The last observed pathogen was Puccinia sorghi which was identified by urediniospore (Figure 4f), because teliospores are generally produced late in the season or not at all. Microscopic structures were developing on both the upper and lower leaf surfaces.

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This year on locality Žabčice, pathogens had a more frequent rate of occurrence (Table 1), than in year 2018 when only two pathogens were observed: Colletotrichum sublineolum and Ramularia sorghi (Nováková and Šafránková 2018). This was probably due to unevenly distribution of precipitation during the vegetation. This fact had a negative effect on the development of fungal pathogens, probably. According to Kuthan (2012), fungal pathogens that have been observed this year have a long history in our country, it can be assumed that the higher intensity cultivation of sorghum under south Moravian condition will lead to higher intensity of sorghum infestation by harmful organisms. As no pesticide product has been registered in the Czech Republic yet, it is necessary for farmers to choose varieties that show higher resistance to fungal organisms and buy only certified seed from verified distributors.

CONCLUSION Sorghum seems to be very suitable crop for semi-arid regions of dry land with unpredictable rainfall. Sorghum can be used in these areas as a suitable replacement for maize, because it has higher resistance to drought and high temperature, which have positive influence on resistance of fungal pathogens, but with the risk of slowly growing on starting vegetation. The most of sorghum fungal pathogens are exotic in nature in our region and they do not have optimal conditions for this development in our geographical conditions, some pathogens expanded over several years, so it is necessary to address this issue. It is necessary to choose a suitable soil location and treatment for the implementation of this crop into crop rotation, more importantly to take into account the pre- crop and, last but not least, a suitable variety that is resistant to the most important pathogens. Multiple observations are necessary to determine the spectrum and importance of pathogens. However, it can be said that the above-mentioned species occur regularly in the Czech Republic conditions. These pathogens should have more influence on quality of cattle feed in following next years.

ACKNOWLEDGEMENTS The paper is an outcome of the project: AF-IGA-2018-tym001: Comparison of the impact of climate change on photosynthesis C3 and C4 plants cycles which are used in livestock feed.

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