**1. Introduction**

Successful crop production depends on many factors, such as crop type [1], variety [2], and environmental factors [3], including climatic conditions [4], soil [5], and water [6]. Biotic and abiotic stresses also affect yield [7]. Direct protection against abiotic factors such as drought [8], salinity [9], extreme cold or heat [10], heavy metal stress [11], and water deficiency or excess [12] is achieved through resistance. However, biotic stresses are also important [13]. The use of pesticides protects against diseases, pests, and weeds (which cause great economic losses) [14,15]. Their use is disadvantageous because it contributes to environmental pollution [16]. In addition, resistance breeding is widely used to reduce the use

**Citation:** Sz˝oke, L.; Moloi, M.J.; Kaczur, D.; Radócz, L.; Tóth, B. Examination of Different Sporidium Numbers of *Ustilago maydis* Infection on Two Hungarian Sweet Corn Hybrids' Characteristics at Vegetative and Generative Stages. *Life* **2023**, *13*, 433. https://doi.org/10.3390/ life13020433

Academic Editors: Wajid Zaman and Hakim Manghwar

Received: 5 January 2023 Revised: 30 January 2023 Accepted: 31 January 2023 Published: 3 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

of pesticides. Plants have a specific defense system that is activated after infection [17]. The cuticle is the plants' first defense against fungal invasion [18]. The structure of the cuticle can be different in different plant species [19] and its effectiveness against pathogens is also different [20]. When the fungal invasion is successful, plants produce secondary metabolites to protect themselves [21]. Phenolic terpenes and nitrogen/sulfur-containing compounds are synthesized in plants [22]. The roles of phenolic compounds against pathogens are well studied [23]. For example, benzaldehyde (against *Botrytis cinerea*, [24]), protocatechuic acid (against *Colletotrichum circinans*, [25]), salicylic acid (against *Eutypa lata*, [26]), vanillic acid (against *Phytophthora infestans*, [27]), chlorogenic acid (against *Fusarium osysporum*, [28]), naringin (against *Penicillium digitatum*, [29]), flavones (against *Aspergillus*, [30]), oleuropein (against *Phytophthora*, [31]), Nobiletin (against *Phoma tracheophyta*, [32]), Geinstein (*Monilinia fructicola*, [33]), and Hordatin A (against *Helminthosporium sativum*, [34]). Other important molecules in the plant-pathogen interaction are the reactive oxygen species (ROS) [35]. After infection, plant cells produce ROS such as hydrogen peroxide (H2O2), singlet oxygen ( 1O2), superoxide anions (O2 −), and hydroxyl radicals (−OH) [36]. ROS can be important molecules for signal transduction (at low concentration) or toxic (at high concentration) for plants [37]. One of the most detrimental effects of ROS is the induction of lipid peroxidation in the cell membrane [38]. ROS are mainly produced in peroxisomes [39], mitochondria [40], and chloroplasts [41]), which can interfere with metabolic processes. To eliminate ROS, plants use antioxidant enzymes [42]. Ascorbate peroxidase (APX) degrades H2O2 to water using ascorbic acid as a substrate. Several APX isoforms in plants are distributed in different cellular compartments such as chloroplasts, mitochondria, peroxisomes, and cytosol [43]. Superoxide dismutase (SOD) catalyzes the dismutation of the superoxide anion radical (O2 −) to water and H2O2. Their classification is based on their subcellular location and bound metal cofactor (Cu/Zn, Mn, Fe, and Ni) [44]. Guaiacol peroxidase (POX) is essential for lignin biosynthesis and neutralizes H2O2 [45]. Corn smut is a major corn pathogen capable of infecting corn at vegetative and generative stages, leading to serious yield losses [46], especially in sweet corn, which has a great economic impact [47]. Corn smut is a biotrophic pathogen, which causes galls on all aerial parts of its host plants but does not cause the death of cells. To evaluate the impact of a biotrophic pathogen on its host plants, many physiological, morphological, biochemical, and quality parameters give an understanding of the impacts of pathogen infection. Biotrophic pathogens live and complete their life cycle in the host plants. To survive, they derive nutrients from the host plant, leading to reduced growth [48]. Corn smut infection has a significant impact on the leaf chlorophyll content, which can be measured using different methods. During corn smut infection, chlorosis would appear 3–5 days after infection, which is an indication of chlorophyll loss [49]. Therefore, measurements of chlorophyll content are valuable. Infection may also cause oxidative stress in plants [50,51]. The oxidative burst in host plants may activate the antioxidative mechanisms. Sz˝oke et al. [52] showed that the corn smut infection increased the activities of antioxidant enzymes (SOD, APX, and POX) and the malondialdehyde (MDA) content in the infected fodder and sweet corn hybrids.

Corn smut also has a significant impact on sweet corn yield. Clough et al. [53] found a strong correlation between the intensity of the corn smut infection and kernel characteristics. They stated that when the gall size was bigger, fresh weight, length, diameter, and kernel depth were smaller. According to Pál-Fám et al. [54], corn smut infection significantly decreased the ear, grain and cob weights of the fodder corn, causing significant economic damage. Moreover, they stated that the corn smut-infected cob had lower dry matter, fiber, and ash contents. Keszthelyi et al. [55] also stated that the corn smut infection reduced the dry matter, protein, fat, fiber, and ash contents of the fodder corn.

The effects of different diseases on the nutrient content of the host plant have also been reported. A high concentration of the corn smut infection increased the amounts of Fe and Zn in the shoots and roots of infected plants [56]. The *Candidatus phytoplasma* L. asiaticus infection decreased the N and P contents of citrus species [57]. The Fusarium-infected tomato plants had a lower Cu content compared to uninfected control plants [58]. Mineral nutrients have an important function in the interaction between plants and pathogens [59]. The element contents of crop plants may differ depending on the host plant and the type of plant pathogen [60].

Sz˝oke et al. [52] showed that infection of sweet corn at the vegetative stage with a high amount of corn smut sporidia (10,000 sporidia/mL), under controlled conditions in the greenhouse negatively impacted the photosynthesis pigments and growth parameters. They suggested a follow-up study to establish the effects of corn smut at lower loadings. During the experiment, the two infection periods were simulated that are the most typical in corn cultivation. The first peak of infection mostly affects young corn plants, which are most characteristic during the period of development of mechanical damage caused by the frit fly or mechanical inter-row weeding cultivation. Another such period is the stage of the emergence of young corn cob initiation. The different sporidia concentrations are a good representation of the level of infection pressure, which may be related to the cultivation variants. The physiological changes that occur as a result of the infection are a good representation of the defense reactions and physiological changes of the individual corn plants. These changes in plant physiology are not only correlated with the amount of infectious sporidia material but also strongly depend on the type of crop (for example, sweet or fodder corn) and its phyto-phenological state of development. Therefore, the current research examined the effects of different concentrations (2500, 5000, and 10,000 sporidia/mL) of corn smut inoculum, on different morphological (plant height and stem diameter) and biochemical parameters (chlorophyll, protein, and MDA contents; the activities of SOD, APX, and POX) at the vegetative (V4–V5) and generative (V7) stages. The corn smut attacks the embryonic tissue when the tissue is already in the differentiation phase and smut is not able to infect during this stage, meaning that there will be no tumor formation.

The goal for including different infection times, which was not established by Sz˝oke et al. [52], was to examine if there are any other roles of corn smut infection besides tumor formation (e.g., effects on quality and quantity). In addition, monosporidial inoculation does not cause tumor formation but has negative impacts on plant growth and several other physiological processes. At the V7 stage, measurements focused on cob parameters like cob length and diameter, kernel weight, 100 grains fresh and dry weight, element content of grains, and the quality characteristics (dry matter, fiber, fat, ash, nitrogen, and protein). The first goal of this study was to examine the tumor formation at the V4–V5 phenological stage because the first hypothesis was that there is no tumor formation at low (2500 sporidia/mL) inoculation, The second hypothesis of this research was that the corn smut infection negatively affects the morphological, physiological, biochemical, and quality parameters, as well as the element content and other quality characteristics of grains irrespective of the lower dosage. The third hypothesis was that the 10,000 sporidia/mL has more negative impacts on the measured parameters relative to the 2500 and 5000 sporidia/mL treatments. Furthermore, the goal was to examine which phenological stage (V4–5 or V7) is more susceptible to the corn smut infection.

### **2. Materials and Methods**
