Identification and Pathogenicity of the Soybean Root Rot Pathogen in Arid Conditions

: Soy is an important oilseed crop in Kazakhstan. However, its yield is significantly reduced due to an increase in the development of fungal diseases with soil infection. This study aims to identify the root rot pathogen in soybean varieties in Western Kazakhstan and assess its pathogenicity. Samples of soybean roots to determine the root rot pathogen were selected at the experimental plots of the Aktobe agricultural experimental station in 2021. Genetic analysis of isolates observed on potato dextrose agar medium from soybean root slices was carried out using the sequencing reaction of internal transcribed spacer region nucleotide sequences using the Big Dye Terminator protocol (applied biosystems) with a further comparison of data with the Gen bank database. A test for the pathogenicity of the identified isolates of the fungus was carried out. Statistical data processing was carried out using the R-studio software according to the nonparametric Kruskal Wallis test. Phylogenetic analysis of samples of soybean affected by root rot showed that the main biotic agent causing root rot was the imperfect fungus Fusarium equiseti . Sequencing of the highly informative internal transcribed spacer region of fungal isolates allowed identifying strains 1 and 2 belonging to the species Fusarium equiseti. The pathogenicity test established that isolates of the Fusarium equiseti fungus caused discoloration of seedlings and plant roots and necrosis in soybean seedlings


Introduction
Kazakhstan is an agrarian country with huge areas of land used for growing crops and animal husbandry (Kashina et al., 2022). Soy is one of the important agronomic crops attracting increasing attention in Kazakhstan, due to its growing demand as an oilseed crop and animal feed to ensure food security (Makulbekova et al., 2017;Kuldybayev et al., 2021;Suleimenova et al., 2021). The global experience of successful crop cultivation implies seasonal crop alternation, i.e., rotation with other grain or feed crops (Rodriguez et al., 2021;Bome et al., 2022), as well as the use of cover crops (Wen et al., 2017) to avoid the emergence of fungal and bacterial diseases transmitted through the soil. Fungal diseases, under favorable conditions, can significantly reduce the yields of important crops (Karakotov et al., 2020). Thus, in Brazil, the world leader in soybean production (121.8 million Metric Tons (MT), food and agriculture organization corporate statistical database (FAOSTAT, 2020), due to rust that affected 90% of the country's territory, crop losses in only two states in 2003 amounted to 2.2 million tons (Yorinori et al., 2005). In the USA, the average annual crop losses due to diseases are estimated at 11% of total production (Mueller et al., 2016;Dutbayev et al., 2006), where the soy nematode (heterodera glycines ichinohe) accounts for more than 30% of crop losses, which can occur without noticeable above ground symptoms (Mueller et al., 2016). Argentina, the third largest producer, and exporter with 6.9 million tons (Gale et al., 2019), is also significantly 203 affected by soil pathogens and especially oomycetes, which results in crop losses of up to 20% (Lerch-Olson and Robertson, 2020). Among the most common diseases, germination diseases, sclerotiniosis stem blight, and phytophthora root rot are the most characteristic ones of the top soybean producers in Canada (Lerch-Olson and Robertson, 2020) and China (Yang et al., 2020). In turn, Fusarium damping off and root rot, Alternaria leaf spot, seed rot, and anthracnose are more often mentioned in the soybean regions of Ukraine (Mykhalska et al., 2019), Russia (Kurilova, 2010) and India (Nataraj et al., 2020). The above-mentioned rust and charcoal rot are most often noted in Bolivia (Yorinori et al., 2005) and Paraguay (Orrego Fuente et al., 2009).
Soybeans experience biotic stress from extensive exposure to Fusarium pathogens affecting their seeds, seedlings, leaves, and roots (Bienapfl, 2011). Symptoms of soybean Fusarium all over the world are the death of the growth point, leaf spot, and bean and seed rot, but the most common ones are root rot and tracheomycosis related plant damping off (Kurilova, 2010).
The purpose of our study was to identify the root rot pathogen in European soybean varieties in Western Kazakhstan using modern Deoxyribonucleic Acid (DNA) sequencing techniques and to assess its pathogenicity.

Materials and Methods
Samples of soybean roots affected by root rot were taken at the Aktobe agricultural experimental station, in Western Kazakhstan (50°21ʹ22.2ʺ N, 57°23ʹ47.2ʺ E). Twelve soybean varieties from the Eurasian collections were examined for the presence of root rot: Samer 1, Samer 2, Samer 3, Samer 5, Svapa, Tanais, Belor (Russia), Toury (Czech Republic), Isidor (France), Maple Ridge (Canada), Anastasiya, Cheremosh (Ukraine). Root samples were taken during soybean ripening at the end of August 2021 to determine the impact of the disease on crop yields.
One hundred and twenty samples of soy roots of each variety with a size of 1  1 cm were collected randomly. To obtain the Fusarium fungus inoculate, these soybean samples were disinfected by washing in 70% ethanol for 10 sec, followed by immersion in a 1% solution of sodium hypochlorite for 3 min. According to the methods developed by the Kazakh institute of plant protection and quarantine, after three rinses in sterile water, the roots were placed in aseptic conditions in wet boxes on sterile premoistened filter paper. The wet boxes (plastic containers), in turn, were sterilized with a 95% ethanol solution. Under these conditions, the roots were incubated at 25°C in the dark until the fungal mycelium was well expressed. The formed conidia of the fungus were used to isolate the DNA. Pure Fusarium fungus cultures were obtained by re-seeding the initial culture with four varieties for 7-9 days on Potato Dextrose Agar (PDA) medium under thermostat conditions at a temperature of 25°C (Dutbayev et al., 2006).
Three-seven daily strains of fungi were used in the study. The mycelium was frozen at 20°C. Then it was ground with a pestle in a 1.5 mL Eppendorf test tube to a powdery state. After grinding, fungi samples with a pure culture were transferred for further sequencing. The DNA concentration was determined on a double strained DNA (dsDNA) High Sensitivity (HS) scale. After amplification, the resulting Polymerase Chain Reaction (PCR) product was purified. The universal primers of the fungi Internal Transcribed Spacer (ITS) region were used in the study.
Phylogenetic analysis of DNA sequences was performed using MEGA6 software to compare the regularities of the molecular evolution of genes through a graphical interface in real time (Tamura et al., 2013). The alignment of nucleotide sequences was carried out using the ClustalW algorithm as an operational generation of the tree and its sequential process (Li, 2003). The neighbor joining method was used to restore the correct phylogeny (Elias and Lagergren, 2005).
To understand the pathogenic nature of the isolated and identified Fusarium isolates, a pathogenicity test was conducted using three soybean varieties characterized by the lowest, average, and severe susceptibility to root rot (Cummings et al., 2018;Dutbayev et al., 2021).
Statistical data processing was carried out using the R studio software according to the nonparametric Kruskal Wallis test. The significance of the calculations was evaluated using the p-value (Aphalo, 2017).

Results and Discussion
Pathogenic fungi of the Fusarium genus are very widely represented in all cultivated plants of the agricultural sector. The morpho structural features of many groups are quite similar and cause some difficulty in their morphological identification . The emerging need for rapid and accurate diagnosis of fungal species was successfully solved by molecular identification of nucleotide sequences based on PCR technology (Raja et al., 2017). In recent years, the diagnosis using the recombinant DNA (rDNA) ITS region has found the greatest use for this identification of fungi, as it determines the sequence of nucleotides in the segment between two large and small subunits of rRNA genes (Tedersoo et al., 2015), although according to Nilsson et al. (2019), the ITS method has several limitations, such as the presence of polymorphism and low-quality sequences. At the same time, there are also highly informative identification methods, such as Translation Elongation Factor 1-alpha (TEF-1α) and β-Tubulin (BT), which overcome the ITS limitations and are recommended as secondary barcoding for the vast majority of Ascomycota genera (Dutbayev et al., 2022). Nevertheless, Zhou et al. (2018)

extracted 102
Fusarium isolates from infected soybean seedlings in Canada, of which 10 species of fungi of the Fusarium genus were identified благодаря the joint use of sequencing methods based on ITS and EF-1α regions. Pure cultures of Fusarium fungi were obtained after the incubation of soybean root samples on PDA under thermostat conditions for 9 days (Fig. 1).
DNA concentrations measured in ng/µl according to the Qubit 2.0 fluorimeter readings based on previously and further crushed isolate samples are shown in Table 1.
As a result of amplification with its primers, PCR products with a size of about 550 bp were obtained.
As a result of cleaning, we obtained the pronounced PCR products shown in Fig. 2 that corresponded to the required purity. The concentration of the purified product is shown in Table 2.
Based on the capillary foresis of the PCR product after the second purification, the clarity, and purity of the peaks in Fig. 3, indicate the absence of cross contamination of cultures and the good quality of the analysis.
A phylogenetic tree was constructed with the closest related strains (Fig. 4), which allowed taxonomic identification of the studied strains.
Taxonomic identification of strain 1 shows that isolate MK562068 Fusarium equiseti 09 is the closest related strain. The degree of homology was 100%, which shows that the studied strain can be attributed to this species. Taxonomic identification of strain 2 (Fig. 5) shows that isolate MK562068 Fusarium equiseti 09 is the closest related strain. The degree of homology was 100%, which shows that the studied strain can be attributed to this species.

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Another representative of this genus was also identified in the studied mixture of Fusarium sp fungus isolate. GAGGTCACATTCAGAAGTTGGGGGTTTAACG  GGTTGGCCGCGCCGCGTTCCAGTTGCGAGGGGT  TAGCTACTACGCAATGGAGGCTGCAGCGAGACC  GCCACTAGATTTCGGGGCCGGGCTGGAGTACAG  CCCGATCCCCAACACCAAACCCGGGGGCTTGAG  GGTTGAAATGACGCTCGAACAGGCATGCCCGCC  AGAATACTGGTGGGCGCAATGTGCGTTCAAAGA  TTCCATGATTCACTGAATTCTGCAATTCACATTA  CTTATCGCATTTTGCTGCGTTCTTCATCGATGCC  AGAACCAAGAGATCCGTTGTTGAAAGTTTTGAT  TTATTTGTTTTTTAGACTCACAAGTTACACTAAA  ATCAGAGTTTTGTGGTTCCTGCGGCGGGCCGTCC  CGTTTTACGGGGCGCGGGCTGATCCGCCGAGGC  AACATAAAGGTATGTTCACAGGGGTTTGGGAGT  TGTAAACTC. According to Fig. 6, it can be determined that MG267121.1:25-497 Fusarium brachygibbosum is the closest related strain of the species. The degree of homology was 100%, which shows that the studied strain can be attributed to this species.
Simultaneously isolated isolate of the fungus Fusarium brachygibbosum is characterized by a similar white color and abundant aerial mycelium, like Fusarium equiseti. However, it differs from the latter by the presence of red pigmentation in the middle of the isolated colony. In turn, the central part of the mycelium in isolates of Fusarium equiseti 09 was predominantly light yellow.
A similar coloration of Fusarium brachygibbosum isolates was previously noted by Wang et al. (2021).
In the process of field and laboratory studies, based on the visual symptoms of the roots and the presence of a colony of fungi during their initial isolation, we determined that the varieties Maple Ridge, Samer 5, and cheremosh were more often susceptible to fungal diseases. In the varieties as Samer 1, Svapa, and Toury, the lowest fungal presence was detected, thus characterizing the latter as the most resistant to pathogens.
An earlier study conducted in the Caspian water area by Khapilina et al. (2011) isolated from the soil and identified an isolate of the F. sporotrichioides species by using the Integrated Genome Sizing (IGS) sequencing method of the DNA region. At the same time, in the Almaty region, the work of Ignatova et al. (2013) showed the common presence of Fusarium fungi such as F. equiseti and F. oxysporum in the soil of such crops as alfalfa, rapeseed, esparcet, mille, and soybean. In general, the southeastern region, known as favorable for soybean growth, is characterized not only by the presence of the representatives of Fusarium, Penicillium, and Aspergillus genera but also by pathogens such as Cercospora sojina, Cercospora kikuchii and Septoria glycines (Zatybekov et al., 2018). Soybeans, which grow mainly in the warm southern regions, are now actively cultivated in the northern and eastern regions of Kazakhstan, representing the most adapted varieties with a short growing season (Abugalieva et al., 2016). There is practically no information on these or other pathogens typical for legumes from western Kazakhstan.
In the last decade, the technique of DNA sequencing by the ITS region has been widely used in phytopathology as a relatively fast and reliable method for the procedure of identifying microorganisms (Parikh et al., 2018). Thanks to this technology, the obtained nucleotide sequence of isolates according to the comparisons made with the Gen bank database and phylogenetic tree alignments allowed us to identify the isolate as Fusarium equiseti.
Determination of the infectious nature of diseases in leguminous crops is of paramount importance in Kazakhstan as an initial level of pathogen control. In the last decade, the use of genetic methods at the sites of the southeastern, eastern, and northern regions of Kazakhstan has allowed Kazakh specialists to genotype this material, including for the identification of DNA markers of resistance to certain fungal diseases, identifying genes or Quantitative Trait Loci (QTL) associated with disease resistance, which is one of the most effective preventive measures to overcome biotic dependence. In this case, they identified the QTL associated with soybean productivity and resistance to Fusarium, cercosporosis, septoria, and purpurea cercosporosis (Zatybekov et al., 2018). Moreover, to adapt the culture to the arid conditions of western Kazakhstan, the influence of some species of fungi of the Fusarium genus on photosynthesis, plant productivity, and appropriate measures to combat pathogens was clarified . Works confirming the positive effect of a mixture of fungi or a combination of fungal and bacterial isolates have acquired special ecological significance as measures to combat such soybean diseases as root and stem rot Zhang et al., 2013). This fact of preference for the use of biocontrol agents in the regulation of pathogenic organisms in both soybeans and other crops is beyond doubt. This is especially relevant due to the extreme limitations of the studies conducted on legumes in Kazakhstan concerning the adaptation of the crop to various environmental conditions of the country against the background of its increasing export and internal demand from year to year (Makulbekova et al., 2017).
According to the pathogenicity test and Koch's Postulate, repeated isolation and identification of the isolate, F. equiseti 09 caused discoloration of seedlings and plant roots and necrosis (Table 3). The highest values of the seedlings damping off, the spread, and the development of the disease was noted in the Maple Ridge variety (80.0, 84.0, and 71.0%, respectively) corresponding to a susceptible variety to Fusarium root rot, and the lowest values were noted in the Toury variety (15.0, 23.0 and 14.0%, respectively). The Toury variety is classified as resistant to root rot. In turn, the symptoms of the disease did not appear on the control soybean seedlings. Summarizing the study, it can be assumed that the F. equiseti 09 isolate is of soil origin. However, this conclusion can be fully confirmed only after an infectious analysis of the seeds of the culture.   It was previously reported (Chittem et al., 2015) in the study of field pea roots that F. equiseti (6.5%) and F. redolens (6.8%) were weakly pathogenic species. In turn, in the work of Japanese experts (Horinouchi et al., 2007), it was noted that Fusarium equiseti was the most effective organism for controlling and reducing the stem and root rot of tomatoes caused by Fusarium oxysporum f. sp. radicis-lycopersici. Thus, as a result of the pathogenicity test, it was found that isolates of the Fusarium equiseti fungus could cause discoloration of seedlings and plant roots and necrosis in soybean seedlings.

Conclusion
The purpose of our study was to identify the root rot pathogen in soybean varieties in Western Kazakhstan and assess its pathogenicity. Sequencing of its region of fungi isolated from soybean roots identified strains 1 and 2 belonging to the Fusarium equiseti species. The presence of Fusarium brachygibbosum fungus was also detected in small concentrations. As a result of the pathogenicity test, it was found that isolates of the Fusarium equiseti fungus could cause discoloration of seedlings and plant roots and necrosis in soybean seedlings.