Mycobiome of sunflower rhizosphere in organic farming

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Introduction

The rhizosphere soil of plants is a dynamic multi-component ecosystem in which beneficial, neutral, and harmful microorganisms actively function (Ellanska et al., 2017). Cultivated plants are in constant interaction with symbiotic and pathogenic microorganisms in soil biota (Broeckling et al., 2008). Microbial cenosis plays a vital role in the plant communities' functioning, influencing their growth, development, and physiological parameters. The vast majority of soil microbiota increases soil fertility and crop productivity. Pathogenic microorganisms colonizing the rhizosphere soil lead to diseases of cultivated plants (Mendes et al., 2013). Crop yield losses only from root rot caused by phytopathogenic fungi are 5-15%. However, they can reach a much higher level (40-70%) (Antonyak et al., 2013). Thus, the rhizosphere soil of cultivated plants is the most critical factor that regulates the phytosanitary condition of agrocenoses and determines the infectious potential of the soil-plant-pathogen-microbiota system (Kopylov, 2012).

Continuous oilseeds cultivation for a long time leads to the rhizosphere soil microbiome impoverishment in agro phytocoenoses due to reduced species microbial biodiversity. With the help of the literature analysis, it was established that due to the increase in phytopathogenic micromycetes number, the infectious load on other crops in crop rotation increases (Petrenkova, 2014). This significantly affects the processes that occur in the rhizosphere and the tissues of host plants and inhibits their growth and development, reducing yields (Kirichenko et al., 2010; Petrenkova et al., 2012). Necrotrophic phytopathogens, including fungi of the genera Aspergillus spp., Penicillium spp., Fusarium spp., Alternaria spp, the leading producers of mycotoxins that parasitize sunflower plants during the growing season (Kirichenko et al., 2010). Some stages of their development occur in the soil during interaction with host plants (Antonyak et al., 2013).

Physiologically active plant substances of different varieties and crop hybrids significantly affect the structure and functioning of microbial populations in the rhizosphere soil (Antonyak et al., 2013; Kostyuchenko, 2014; Sugiyama, 2019), where metabolites are exchanged between cultivated plants and microorganisms (Ellanska et al., 2017).

Sunflower is well known for its allelopathic potential. More than 200 natural allelopathic sunflower compounds, including heliannuols, terpenoids, flavonoids, chlorogenic acid, isochlorogenic acid, and scopolin, are characterized by inhibitory or stimulating effects against pathogenic microorganisms and segetal vegetation (Jabran, 2017).

Currently, considerable attention is paid to the study of crop interaction with the rhizosphere soil microbiome. It is known that the cultivated plant vegetative organs are a source of microbial biodiversity in the rhizosphere soil (Bruinsma et al., 2003). However, the influence mechanism of variety/hybrid on the number and microorganisms species composition has not been studied enough.

Therefore, our research was aimed at determining the influence of sunflower hybrids on the micromycetes number and species composition in rhizosphere soils in organic production.

Materials and Methods

The research was conducted during 2018-2020 based on the laboratory of agroecosystems and organic production biocontrol at the Institute of Agroecology and Nature Management of NAAS, Ukraine. Samples of rhizosphere soil were taken in the fields of the Skvyra research station of organic production of Institute of Agroecology and Environmental Management NAAS of Ukraine by the envelope method on the diagonal of the field by DSTU 4287: 2004. The number of rhizosphere microorganisms of Dushko and Oliver sunflower hybrids was determined by budding, flowering, and physiological maturity. Soil type - low humus coarse-grained medium loam black soil.

The territory of Skvyra Research Station is characterized by a moderately warm, moderately humid climate, which is conducive to the growth and development of sunflower plants. It is well known that the ontogenesis of sunflower and the spread and development of diseases are significantly affected by temperature and rainfall. The integrated indicator of these factors is the hydrothermal coefficient (HHC, Selyaninov coefficient).

Sunflower grows well for an HHC of 1.0-1.5 (sufficient hydration). However, at HHC ≥1.0, the probability of culture damage by various diseases, mainly white and grey rot, increases sharply. Only specific disease development is possible for smaller hydration, except for theAlternaria, dry head rot, verticillium wilt, Orobanche cumana (sunflower broomrape) (Dermenko, 2017).

According to the results of the calculation of HHC, presented in Table 1, we can conclude that the growing season in 2018 was characterized as sufficiently wet (HHC 1.35). At the same time, the vegetation period of 2019 was arid, the drought was not intense (HHC 0.9), and 2020 was sufficiently moist (HHC 1.0).

Table 1. The value of the hydrothermal coefficient (HHC) during the growing seasons.

However, meteorological conditions during the research period, namely: high air temperature and high rainfall during the growing season, significantly impacted the formation of micromycetes in the sunflower agrocenosis.

Protection of sunflower crops from diseases was carried out according to the scheme presented in Table 2.

Table 2. Scheme of sunflower crops protection from diseases (2018-2020)

The number of micromycete populations in the rhizosphere was studied using dilution and surface sowing of soil suspension on nutrient media (DSTU 7847:2015, 2016). Chapek's medium was used for the study. The number of micromycetes was expressed in colony-forming units (CFU) in 1 g of dry soil. The number of microorganisms in 1 g of dry soil was determined by the formula (1):

(1) (DSTU 7847: 2015, 2016)

where N is the number of cells or colony-forming units (CFU) in 1 g of the studied soil;

a is the average number of colonies grown in the cup of this dilution;

n - breeding number from which the crop was made;

V- is the volume of soil suspension taken for sowing, 0.1 cm3.

The number of microorganisms in 1 g of dry soil is calculated by the formula (2):

x = N * K (2)

where x is the number of cells or colony-forming units (CFU) in 1 g of dry soil;

N is the number of cells or colony-forming units (CFU) in 1 g of the studied soil;

K-conversion factor to dry soil.

For accurate calculations of the number of microorganisms in the soil, its humidity was determined in advance following DSTU ISO 11465. The count of the number of colonies of micromycetes in Petri dishes was performed using an automatic counter SCAN4000 (Interscience, France). Identification of isolates of microscopic fungi to genus and species was carried out on a biological microscope DN-200D by determinants (Litvinov, 1967; Pidoplichko, 1972), using an online database "MycoBank".

The rate of occurrence (RO) of phytopathogenic micromycetes in the rhizosphere soil of sunflower was determined by the formula (3) (Mirchyk, 1988):

Where A is the frequency of occurrence of species;

B-the number of samples in which these species were selected;

C-the total number of selected species.

Statistical processing of experimental data was performed using statistical and correlation methods of mathematical statistics using Microsoft Excel software.

Results

We revealed that the number of micromycetes in the rhizosphere is subject to significant fluctuations depending on the sunflower hybrids and the technology of their cultivation. The number of micromycetes in the rhizosphere of sunflower hybrids in the conditions of traditional technology with the use of chemical fungicides, depending on the hybrid, ranged from 5.1 to 6.3 thousand CFU/g of dry soil (Figure 1A). The rhizosphere soil of Dushko hybrid plants in the budding phase amounted to 5.9 thousand CFU/g of soil. In the flowering phase, there was a slight decrease (up to 5.6 thousand CFU/g of soil) in the number of micromycetes. During the physiological maturity phase of the rhizosphere plant of the Dushko hybrid, this indicator increased rapidly and reached an average of 6.3 thousand CFU/g of dry soil.

Figure 1: The number of micromycetes in the rhizosphere soil of sunflower plants against the background of different technologies for growing crops.

The number of micromycetes in the rhizosphere soil of Oliver sunflower in the budding phase was slightly lower compared to the Dushko hybrid and amounted to 5.4 thousand CFU/g of soil. However, in the Oliver hybrid rhizosphere, in the flowering phase, the number of micromycetes increased significantly and averaged 6.1 thousand CFU/g, and in the phase of physiological maturity, it decreased rapidly and amounted to 5.1 thousand CFU/g of dry soil (Figure 1A).

Thus, it was found that against the background of traditional technology during the plants' ontogenesis of the studied sunflower hybrids, the number of micromycetes in the rhizosphere soil varies depending on the hybrid and the phase of plant development.

During organic sunflower production against the background of biological remedies, the number of micromycetes in the rhizosphere soil of sunflower plants of the studied hybrids ranged from 5.4 to 6.7 thousand CFU/g of dry soil (Figure 1B). Thus, the number of CFU micromycetes in the Dushko sunflower hybrid rhizosphere soil in the budding phase was 6.2 thousand CFU/g of dry soil. Simultaneously, in the flowering phase, there was a significant decrease in the density of micromycetes (up to 5.4 thousand CFU/g). In the physiological maturity phase, there was a significant increase in the number of micromycetes in the rhizosphere of the Dushko sunflower hybrid (up to 6.4 thousand CFU/g).

A significant decrease in the number of CFU micromycetes during plant ontogenesis under organic production conditions was observed in the rhizosphere soil of Oliver sunflower hybrid. Thus, if the number of micromycetes in the rhizosphere of plants of the studied hybrid in the budding phase was 6.7 thousand CFU/g of dry soil, then in the flowering phase, the number was 0.4 thousand CFU lower. In the phase of physiological maturity, the number of micromycetes in the rhizosphere of the Oliver hybrid continued to decrease to 5.5 thousand CFU/g of dry soil (Figure 1B). The obtained results may indicate a significant pressure of the root exometabolites of Oliver sunflower plants on the population of micromycetes in the rhizosphere.

Simultaneously, the number of micromycetes in the rhizosphere soil of the studied hybrids was correlated with the value of HTC during the plant vegetation period for 2018-2020. (Table 3).

Table 3. Correlation indices between the number of micromycetes in the rhizosphere of the studied hybrids and the value of HTC.

Based on the statistical processing result, we found that the micromycetes number in the rhizosphere soil of the studied sunflower hybrids was directly correlated with the value of the HTC of the growing season 2018-2020.

Thus, we determined that the number of phytopathogenic micromycetes differs, indicating a significant effect of root exometabolites of different sunflower hybrids and their cultivation technologies on the micromycetes population in the soil rhizosphere.

The species composition and micromycetes occurrence rate selected from the rhizosphere soil of the studied sunflower hybrids during the growing season were determined. According to the research results, the species composition of micromycetes isolates of rhizosphere Dushko and Oliver sunflower hybrids is represented by fungi of the genera: Aspergillus, Alternaria, Penicillium, Fusarium, and Trichoderma (Table 4).

Table 4. The frequency of micromycetes selected from the rhizosphere soil of sunflower hybrids Dushko and Oliver during the growing season.

Dushko hybrid rhizosphere soil was characterized by the largest species of micromycetes diversity, from which 6 species were isolated (Figures 2-4). Thus, in the rhizosphere soil of this hybrid in the phase, the species composition was represented by genus Aspergillus fungi, which included species A. niger and A. flavus with a frequency of 55% and 50%. Typical representatives of Dushko hybrid rhizosphere soil were fungi of the genus Penicillium, the frequency of which in the budding phase was 60%, and in the phase of physiological maturity - 80%. Simultaneously, the phytopathogenic complex of the sunflower root zone of the studied hybrid formed the fungus A. alternata with a frequency of 50% in the flowering phase and 60% in the physiological maturity phase (Table 4).

Figure 2: Relationship between the number of micromycetes in the sunflower rhizosphere soil and the value of HTC (Dushko hybrid in the conditions of traditional (Figure 1A) and organic (Figure 1B) cultivation technology).

Figure 3: Relationship between the number of micromycetes in the rhizosphere soil of sunflower plants and the value of HTC (Oliver hybrid in the conditions of traditional (Figure 2A) and organic (Figure 2B) cultivation technology).

Figure 4: The spectrum of micromycetes in the rhizosphere soil of sunflower plants of the Dushko hybrid.

A typical representative of the Oliver hybrid rhizosphere soil was the phytopathogenic fungus F. oxysporum, characterized by the different frequency of occurrence during plant ontogenesis (Table 4). Thus, in the budding phase, the frequency of this species occurrence was 20%, in the flowering phase, it increased to 35%, and in the physiological maturity phase, it significantly decreased (up to 10%). It can be assumed that Oliver sunflower hybrid root exometabolites can exert significant pressure on its population in agrocenosis.

In the phase of physiological maturity, not only the species composition of the mycological group changed, but the species diversity of potential phytopathogens expanded due to micromycetes: A. alternata, F. oxysporum, and A. flavus.

As can be seen from studies, in the rhizosphere of sunflower plants, hybrids Dushko and Oliver during the growing season formed a specific micromycetes group, among which the dominant toxin-forming species were fungi of the genera Aspergillus and Penicillium and species with phytopathogenic properties (known as A. alternata, F. oxysporum). Sunflower crops and leads to biological contamination of agrocenoses during the growing season of plants.

The scientific literature analysis shows that biotic environmental factors significantly influence the number and dynamics of the phytopathogenic micromycetes population in agrocenoses. Among them, the leading factor is the selective pressure of varieties and hybrids of cultivated plants. They can influence the qualitative and quantitative indicators of phytopathogenic background in agrocenoses, which significantly impairs the agroecosystem's biosafety (Parfeniuk et al., 2020).

Sunflower Hybrids, characterized by the appropriate structure of morphological and physiological, and biochemical characteristics, are factors in the mycobiome formation of the rhizosphere soil and vegetative organs of plants, its quantitative and qualitative composition. Our results showed that in the organic and traditional technologies of sunflower cultivation, the number of micromycetes depended on the hybrids and technology of their cultivation.

In the rhizosphere of sunflower hybrid plants, a specific group of micromycetes is formed during the growing season, among which toxin-forming fungi species belonging to the genera Aspergillus and Penicillium and the fungus A. alternata dominate.

Conclusion

The micromycetes number in the rhizosphere soil varies significantly depending on the sunflower hybrid and the technology of its cultivation. In the conditions of traditional technology, the number of micromycetes varies from 5.1 to 6.3 thousand CFU/g of dry soil, and in the conditions of organic technology - from 5.4 to 6.7 thousand CFU/g of dry soil. This can be explained by the significant influence of sunflower plants of different hybrids root exometabolites and technologies of their cultivation on the population of micromycetes in the rhizosphere soil.

Fungi of the genera represent the species composition of micromycete isolates in the Dushko and Oliver sunflower hybrids rhizosphere: Aspergillus, Alternaria, Penicillium, Fusarium, and Trichoderma. The budding phase is dominated by fungi of the genus Aspergillus, which are represented by A. niger and A. flavus species with a frequency of 50 - 55%.

In the physiological maturity phase, in the Dushko hybrid rhizosphere, soil fungi of the genus Penicillium parasitize with a frequency of 60-80%. The phytopathogenic complex of the root zone of sunflower plants is formed by phytopathogenic micromycetes of A. alternata and F. oxysporum. Their frequency is 50%. These micromycetes cause significant damage to sunflower crops and lead to biological contamination of agrocenoses during plants' growing season.

References

Antonyak, G.L., Kalinets-Mamchur, Z.I., Dudka, I.O., Babych, N.O. & Panas, N.E. (2013). Mushroom Ecology [Ekologіja gribіv]. Lviv, Ivan Franko LNU. (in Ukrainian)

Broeckling, C.D., Broz, A.K., Bergelson, J., Manter, D.K. & Vivanco, J.M. (2008). Root exudates regulate soil fungal community composition and diversity. Appl. Environ. Microbiol, 74, 738-744.https://doi.org/10.1128/AEM.02188-07

Bruinsma, M., Kowalchuk, G.A., Veen, J.A.(2003). Effects of genetically modified plants on microbial communities and processes in soil. Biology and Fertility of Soils, 37(6), 329-337. https://doi.org/10.1007/s00374-003-0613-6

Dermenko, O.P. (2017). Sunflower diseases: recommendations for diagnosis and protection measures. Kyiv: the National University of Life and Environmental Sciences of Ukraine. (in Ukrainian).

DSTU 4287: 2004. Soil quality: Sampling. [Effective from 2005-07-01]. Kyiv: State Standard of Ukraine (in Ukrainian).

DSTU 7847: 2015. Soil quality: Determination of the number of microorganisms in the soil by sowing on a solid (agar) nutrient medium [Effective from 2015-06-22]. Kyiv: SE "UkrNDNC" (in Ukrainian).

DSTU ISO 11465: 2001. Soil quality: Determination of dry matter and moisture by weight. Gravimetric method (ISO 11465: 1993, IDT) [Effective from 2001-12-28]. Kyiv: State Standard of Ukraine (in Ukrainian).

Ellanska, N.E., Skrypka, G.I., Yunosheva, O.P. (2017). Microbial groups and biological activity of the root-soil of plants Phlox paniculata L. Visnyk of Odessa National University, 22(2), 67-75. https://doi.org/10.18524/2077-1746.2017.2(41).113306 (in Ukrainian).

Jabran, K. (2017). Sunflower allelopathy for weed control. In: K. Jabran, Manipulation of allelopathic crops for weed control. Springer Briefs in Plant Science, Springer International Publishing AG. 77–86. https://doi.org/10.1007/978-3-319-53186-1_9

Kirichenko, V.V., Petrenkova, V.P., Maklyak, K.M., Borovska, I.Yu. (2010). Results of selection of sunflower for resistance to the main pathogens. UA-AN, Inst. Of Plant Breeding. V.Ya. Yurieva, 98, 3-12. (in Ukrainian). https://doi.org/10.30835/2413-7510.2010.70214 

Kopylov E.P. (2012). Soil fungi as a biological factor influencing plants. Agricultural Microbiology, 15–16, 7-28 (in Ukrainian).

Kostyuchenko, N.I. (2014). Influence of crop rotation and variety on soil microbiological indicators of sunflower agrocenoses in the conditions of the southern steppe of Ukraine. Scientific and technical bulletin of IOC NAAS. (in Ukrainian).

Litvinov M.A. (1967). Key to microscopic soil fungi. Leningrad: Nauka. (in Russian).

Mendes, R., Garbeva, P., Raaijmakers, J.M. (2013). The rhizosphere microbiome: significance of plant beneficial plant pathogenic and human pathogenic microorganisms. FEMS Microbiol Rev., 37(5), 634-663.

https://doi.org/10.1111/1574-6976.12028

Mirchyk T.G. (1988). Soil mycology: textbook. Moscow: Moscow State University Publishing House, 220 p. (in Russian).

Parfeniuk A., Mineralova V., Beznosko I., Lishchuk A., Borodai V. & KrutV (2020). Mycobiota of the rhizosphere of raspberry plants (Rubus idaeus L.) under the influence of varieties and new fertilizers in organic production conditions. Agronomy Research, 18(4), 2550-2558. (in Ukrainian). https://doi.org/10.15159/AR.20.182

Petrenkova, V.P. (2014). Status and prospects of sunflower breeding for disease resistance. Proceedings of the international scientific-practical conference. (in Ukrainian).

Petrenkova, V.P., Borovska, I. Yu., Kirichenko, V.V. (2012). Resistance of sunflower to necrotrophic pathogens Institute of Plant Breeding named after V.Y. Yuriev NAAS, KhNAU named after Dokuchaev. (in Ukrainian).

Pidoplichko N. M. (1972). Penicillia (key for identifying species). Kyiv. Naukova Dumka. (in Ukrainian).

Sugiyama, A. (2019). The soybean rhizosphere: Metabolites, microbes, and beyond. Journal of Advanced Research, 19, 67-73. https://doi.org/10.1016/j.jare.2019.03.005