Geographical Correlation and Genetic Diversity of Newly Emerged Races within the Ug99 Lineage of Stem Rust Pathogen, Puccinia graminis f. sp. tritici, in Different Wheat-Producing Areas

Wheat stem rust caused by Puccinia graminis f. sp. tritici is one of the most destructive wheat diseases worldwide. Identifying stem rust races in general, Ug99 lineage particularly, and determining resistance genes are critical goals for disease assessment. Thirty wheat varieties and monogenic lines with major stem rust resistance genes (Sr) were examined here over the course of three succeeding seasons from 2020 to 2022. Fourteen stem rust races have been identified in ten African countries, as well as Central and West Asia and North Africa (CWANA) and ten European countries. The Ug99 group (Clade I) included four races (TTKSK, TTKST, TTKTK, and TTKTT) and was reported in five African countries (Egypt, Kenya, Rwanda, Tanzania, and Uganda) and Iran, but none of the European countries. On the other hand, none of the races in Clade III-B (TTRTF) and Clade IV-B (TKTTF and TTTTF) were found in Egypt. Furthermore, Egyptian races were clustered separately from races identified from other countries, and six races were found only in Egypt, including PKSTC, RKTTH, TKTTC, TTTSK, TCKTC, and TKTTH. Races from Kenya, Tanzania, Uganda, Rwanda, and Iran were all closely associated with one another, according to correlation analysis. However, most races identified from other investigated regions, including Eritrea, Spain, Ethiopia, Morocco, Italy, Poland, Kenya, Tanzania, and Uganda, were adversely linked with Egyptian races. The diagnostic 350 bp long PCR fragment linked with virulence to Sr31, Clement (Sr31), and Brigardier (Sr31) was used to identify the TTKSK (Ug99) race. The identification of the regional associations and genetic diversity of newly emerged races within the Ug99 lineage of P. graminis tritici in Africa, Asia, and Europe is one of the key goals of this study. It will help plant breeders to develop new resistant lines against the virulent races, especially TTKSK (Ug99) and TTTSK. This helps in ensuring global food security in the context of climate change.


Introduction
After maize, wheat is the second most commonly grown grain in the world. Many diseases affect wheat growth and yield production. Rust diseases are the most prevalent threats that affect wheat plants, such as wheat stem rust, which significantly affects wheat leaves and grain yield [1,2]. Stem rust, caused by Puccinia graminis f. sp. tritici, is the most destructive disease that seriously threatens wheat grain production [3][4][5]. Stem rust caused severe problems in Africa, the Middle East, Central and West Asia and North Africa (CWANA), Australia, New Zealand, Europe, and the USA [6].

Evaluation of Thirty Wheat Lines with Stem Rust Resistance Genes at Adult Plant Stage under Field Conditions
Thirty wheat lines with stem rust resistance genes (Sr ' s) were obtained from the International Maize and Wheat Improvement Center (CIMMYT, México-Veracruz, El Batán, Mexico) and National Small Grain Collection (Aberdeen, ID, USA) and evaluated in two Egyptian governorates, Kafrelsheikh and Sharqia, during three successive growing seasons: 2020, 2021, and 2022 ( Table 1). The monogenic lines described in Table 1 were planted in a three-replicate randomized complete block design (RCBD), with each plot consisting of three rows (3 m long and 30 cm apart). Each row received 5 g of the evaluated monogenic line. A spreading area planted with combinations of the extremely sensitive variety, Morocco, encircled the experiment. After the heading stage, disease responses to the stem rust pathogen were recorded as disease severity (percent) was measured in the tested genotypes using the amended Cobb's scale, as proposed by Peterson et al. [16]. Infection types were recorded according to Roelfs et al. [17], i.e., highly resistant (0), resistant (R), moderately resistant (MR), moderately susceptible (MS), and susceptible (S). a Wheat lines with stem rust resistance genes (Sr's) were obtained from the International Maize and Wheat Improvement Center (CIMMYT, Mexico). "Aberdeen" refers to the National Small Grain Collection (Aberdeen, ID, USA), whereas the "numbers" beside it refer to the year of deposit. b Hard Red Winter Wheat Variety.

Stem Rust Samples Identified in Egypt
In the 2020-2022 growing seasons in the Kafrelsheikh and Sharqia governorates, a stem rust survey was carried out annually to collect infected stems displaying the typical symptoms from wheat monogenic lines and types with substantial stem rust resistance genes (Sr). Paper envelopes (6 × 20 cm) were utilized to gather and store the fifty-seven samples that were obtained. Collected samples (infected stems) were placed on top of their envelopes for the whole night in a room-temperature environment in order to minimize humidity within the samples. After that, samples were maintained until the end of the season in desiccators with calcium chloride at 4 • C in a refrigerator [18].

Race Analysis
The highly susceptible variety Morocco (7-day-old seedlings) was inoculated with urediniospores from each sample collection at the stem rust greenhouse located in the Wheat Diseases Department, Plant Pathology Research Institute, Agricultural Research Center, Egypt, and in GRRC in Denmark. The procedure for vaccination was followed as stated by Stakman et al. [18]. To create an initial film of free water on the plants, which is necessary for spores to germinate and for the infection to take hold, the seedling leaves were rubbed gently with tap water between wet fingers, sprayed with water in the incubation chambers, then inoculated by shaking or brushing rusted materials over the plant leaves. Inoculated seedlings were incubated in a secured glass moist chamber in darkness for 16 h at 18-22 • C and 98-100% relative humidity for 24 h to let the rust spores grow and infect the plants. Subsequently, inoculated plants were transferred to be maintained in the greenhouse at 25-28 • C, with 80-90% relative humidity, 16 K Lux light intensity, and a 16 h light, 8 h dark photoperiod. Inside the greenhouse, each sample was transferred to a place designated for it, covered with an acrylic cylinder so that there was no mixing between the samples, and watched until rust pustules appear. To obtain enough urediniospores to inoculate the differential sets, three single pustules from each sample were separated individually before being inoculated onto the seedling of a highly susceptible wheat variety from Morocco. Each pustule infected a single plant (20 Sr , s).
The plant response was determined on 20 lines divided into 5 groups of 4 lines each, as described by Njau et al. [7], with slight modifications. The first group consists of isogenic Sr-lines 5, 21, 9e, 7b; the second group consists of 11, 6, 8a, 9g; the third group consists of 36, 9b, 30, 17; the fourth group consists of 9a, 9d, 10, Tmp; and the fifth group consists of lines 24, 31, 38, McN [20]. Based on the combination of responses from low infection type (L) and high infection type (H) plants, P. graminis coding (Pt-code) for each isolate was defined using capital letters (Table 3).

Disease Evaluation
After 12 days of the formation of pustules on near-isogenic lines (20 Sr ' s), wheat stem rust infection types for all isogenic lines were documented using a standard illness rating scale of 0-4. Stakman et al. [18] revealed the virulence patterns on differential sets based on the low infection types produced by each line in response to infection (infection types 0, 1, and 2 were avirulent, whereas infection types 3 and 4 were virulent).

DNA Extraction and PCR Amplification
DNA was extracted from 0.1g spores collected from pure cultures maintained on susceptible wheat seedlings in our greenhouse using a genomic plant DNA extraction based on Dellaporta et al. [21] method. Three biological replicates per treatment were analyzed. The purity and concentration of the fungal DNA were determined using a Nan-oDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and DNA concentration was adjusted to 50 ng µL −1 for further PCR amplification. The PCR amplifi-cation was performed in a 25 µL reaction mixture using two particular primers (PgtSSR3F: 5 -GGACCAAAACCAGAACCAGA-3 and PgtSSR3R: 5 -CCCACTCCTAATCCTCACGA-3 ) that were designed for Ug99-associated markers [20]. The PCR reaction mixture contained 2.5 µL genomic DNA (50 ng µL −1 ), 1.0 µL of each reverse and forward primers (10 picomols), and 8.0 µL Milli-Q ® -H 2 O. After optimization, DNA amplification was performed using a thermal cycler (Rocorbett-Research, CG1-96). The thermal cycler was programmed for one cycle of denaturation at 94 • C for 5 min followed by 35 cycles of denaturation at 94 • C for 30 s, annealing at 55 • C for 1 min, and extension at 72 • C for 1 min. A final extension step at 72 • C for 10 min was also performed [22]. PCR products were resolved by electrophoresis in 1.2% agarose gel at 100 V/1 h and visualized under UV light following staining with ethidium bromide (500 µL L −1 ). Mid-Range DNA Ladder 100 bp-3 kbp linear sale (Jena Bioscience, Jena, Germany) was used to determine the molecular weight of the tested materials.

Statistical Analysis
Two-way hierarchical cluster analysis (HCA) was used to better understand the relationship between the 14 newly emerged physiological races of P. graminis f. sp. tritici, their geographical distribution, and/or their virulence formula. Briefly, the presence/absence of the 14 races of P. graminis tritici was converted to a binary data matrix (0 = absence and 1 = presence), as well as the virulence formula of the 14 races of P. graminis tritici (0 = avirulent and 1 = virulent) and used for further statistical analysis. Distance and linkage were measured using Ward's minimum variance method [23], with 95% confidence between groups. Moreover, principal component analysis (PCA) was performed, and its associated scatter plots and loading plots were generated using the singular value decomposition (SVD). Furthermore, correlation analysis was computed to measure the strength of the relationship between newly emerged physiological races and their geographical distribution and their virulence formula.
Numbers refer to the disease severity (%) of the stem rust pathogen, P. graminis f. sp. tritici, recorded after the heading stage using the modified Cobb's scale, adopted by Peterson et al. [16], whereas letters refer to the reaction/infection types, i.e., highly resistant (0), resistant (R), moderately resistant (MR), moderately susceptible (MS), and susceptible (S) were recorded according to Roelfs et al. [17]. Tr= Trace.

Survey of Wheat Stem Rust and Race Identification in Egypt
Infected samples were gathered based on the analysis of 30 wheat monogenic lines and types with stem rust resistance genes over the course of the preceding three successive seasons. The Sharqia governorate had the largest number of successfully isolated stem rust samples (36 samples and 20 isolates over the course of three seasons), followed by the Kafrelsheikh governorate (21 samples and 13 isolates over the course of three seasons). Moreover, the largest number of collected samples and successful races were in 2020 (23 samples and 15 isolates), followed by the 2021 (20 samples and 11 isolates) and the 2022 season (14 samples and 7 isolates), as indicated in Table 5. Eight stem rust races were identified during the three seasons in Kafrelsheikh and Sharqia governorates (Table 6). In 2020, seven races were identified, such as PKSTC, RKTTH, TCKTC, TKKTF, TKTTC, TKTTH, and TTKSK. In 2021, six races were identified, i.e., PKSTC, RKTTH, TKKTF, TKTTC, TTKSK, and TTTSK. The most frequent race was TKTTF (50.0%) in the 2021 and 2022 seasons, respectively. In 2022, four races were identified, such as TCKTC, TKTTH, TTKSK, and TTTSK. The most frequent race was TTKSK (50.0%) in this season. On the other hand, the TTKSK race was identified in all seasons, and the new race TTTSK was only detected in Egypt (Figure 2).  Table 2). The Ug99 group (Clade I) included four races (TTKSK, TTKST, TTKTK, and TTKTT) and was reported in five African countries (Egypt, Kenya, Rwanda, Tanzania, and Uganda) and Iran but in none of the European countries ( Figure 3A). On the other hand, none of the races in Clade III-B (TTRTF) and Clade IV-B (TKTTF and TTTTF) were found in Egypt. Furthermore, HCA showed that Egypt was clustered separately from all other countries, and six races were found only in Egypt, including PKSTC, RKTTH, TKTTC, TTTSK, TCKTC, and TKTTH ( Figure 3A). The presence/absence of the 14 races was used to perform principal component analysis (PCA) in all of the areas investigated. Figure 3B,C illustrate the scatter plot and loading plot related with PCA. The PCA-associated scatter plot clearly distinguished Egypt from all other places, which overlapped in the center of the scatter plot ( Figure 3B). It is worth mentioning that PC1 (Eigenvalue = 8.76) and PC2 (Eigenvalue = 5.24) donated 62.54% and 37.46% of the total variance, respectively. Furthermore, the PCA-associated loading plot ( Figure 3C) showed that six races, including PKSTC, RKTTH, TKTTC, TTTSK, TCKTC, and TKTTH, were positively correlated with Egypt, whereas other races were correlated with other countries, in agreement with the HCA analysis.
To better understand the relationships between different races and their geographical distribution, correlation analysis was carried out. Briefly, correlation analysis showed that PKSTC, RKTTH, TKTTC, TTTSK, TCKTC, and TKTTH were highly geographically correlated to each other and slightly correlated with TTKSK, TTKST, TTKTK, and TTKTT, but negatively correlated with TTRTF, TKTTF, and TTTTF ( Figure 3D). Likewise, Kenya, Tanzania, Uganda, Rwanda, and Iran were highly correlated to each other ( Figure 3E). On the other hand, Egypt was negatively correlated with most of the studied locations, such as Eritrea, Spain, Ethiopia, Morocco, Italy, Poland, Kenya, Tanzania, and Uganda, or at least no significant relationship was noticed, such as Tunisia, Denmark, France, and Rwanda ( Figure 3E).
The geographical distribution of TTKSK (represents Clade I [Ug99]), TTTSK (represents the races that are only found in Egypt), and TKKTF (represents Clade IV-E.2, F) physiological races of stem rust pathogen, Puccinia graminis f. sp. tritici, in Africa, Asia, and Europe is shown in Figure 4. While the Ug99 group was mostly reported from African countries, the Clade IV-E.2, F-associated races were mostly reported from Europe, according to the map. It is worth noting that Egypt has been described as a nexus for the three physiological races depicted on the map in Figure 4.

Virulence Formula Refers to the Genetic Diversity between Newly Emerged Races Puccinia graminis tritici
Fourteen stem rust races have been identified from Africa, CW Asia, and Europe in stem rust greenhouse in Egypt and GRRC in Denmark (Table 7 and Figure 5). The most virulent race was TTKTT, which was virulent to 19 monogenic lines, followed by TTTSK, TTKST, TTKTK, and TTTTF, which were virulent to 18 monogenic lines (Table 7). On the other hand, races TCKTC and PKSTC were the lowest virulent races, which were virulent to only 13 and 14 monogenic lines, respectively (Table 7 and Figure 5A). Furthermore, two-way HCA was used to better understand the genetic background of individual physiological races of P. graminis f. sp. tritici and their virulence to 20 monogenic lines. Briefly, the HCA showed that the 14 studied races were separated into five distinct clusters. Cluster I (C-I) included the highly virulent races TTKSK, TTKTK, TTTSK, TTKST, and TTKTT, whereas TTRTF (virulent to 17 lines) and TTTTF (virulent to 18 lines) were clustered together in Cluster II (C-II) ( Figure 5A). On the other hand, the lowest virulent race, TCKTC, was clustered separately in Cluster III (C-III). Likewise, TKTTC, TKTTF, TKTTH, and TKKTF were clustered together within Cluster IV (C-IV), and PKSTC and RKTTC were clustered together within Cluster V (C-V) ( Figure 5A). Moreover, HCA between lines showed that eight lines (Sr5, Sr7b, Sr9g, Sr30, Sr9a, Sr9d, Sr10, and SrMcN) were susceptible to all studied races and clustered together on the right side of the dendrogram. Line Sr24 was the most resistant line (resistant to 12 races), followed by line Sr31 (resistant to 9 races), and line Sr11 (resistant to 7 races) ( Figure 5A).
To measure the strength of the relationship between the newly emerged races and monogenic lines, correlation analysis was carried out. Correlation analysis between physiological races showed a positive association (ranging from low to high) between most races, except TTKST and TTKTT, which showed a low negative correlation with PKSTC, RKTTC, and TKTTC, TKTTD, TKTTF, TTRTF, and TTTTF ( Figure 5D). Likewise, correlation analysis between monogenic lines showed no association between wheat lines and varieties with stem rust resistance genes (Sr), such as lines Sr5, Sr7b, Sr9g, Sr30, Sr9a, Sr9d, Sr10, and SrMcN, and all other study lines. However, a moderate-to-high correlation was observed between lines Sr11, Sr31, Sr38, Sr24, and Sr6 ( Figure 5E).

PCR-Based DNA Markers
The definition of UG99 (TTKSK) was confirmed by molecular markers using specific primers. Urediniospores were collected from the individual pustules of Puccinia graminis on wheat lines and varieties with stem rust resistance genes (Sr); lines Sr24, Sr31, Sr38, Clement (Sr31), Brigardier (Sr31), and Fleming (Sr6, 24, 36, 1RS-Am). Then, the DNA of these samples was isolated and specific primers of Ug99-TTKSK were used. Data illustrated in Figure 6 show that the diagnostic PCR fragment indicating UG99 (TTKSK) race was detected in P. graminis f. sp. tritici isolates from cvs Clement and Brigardier (with Sr31 gene) as a fragment 350 bp. On the other hand, lines Sr24, Sr38, and Fleming (Sr6, Sr24, Sr36, 1RS-Am) were not detected in any fragments with UG99 (TTKSK) at 350 bp. (Figure 6).

Discussion
Historically, stem rust disease has been a serious threat in several wheat-producing regions [9]. More than 20 years ago, race Ug99 of P. graminis f. sp. tritici was first detected in Uganda and identified as TTKSK based on the North American nomenclature [7]. Up to today, approximately 15 variants were identified within the Ug99 lineage. These variants are spreading to several wheat-producing countries in the Eastern African highlands, as well as Zimbabwe, South Africa, Sudan, Yemen, and Iran. Races of the Ug99 lineage pose a threat to wheat productivity and food security [11] due to their capacity to infect almost 90% of the kinds of wheat grown globally. Unfortunately, we know very little regarding the presence of the Ug99 race in Egypt. Although Patpour et al. [13] and Shahin et al. [14] reported the Ug99 race in Egypt, some Egyptian varieties, such as Misr-1 and Misr-2, were chosen from CIMMYT wheat genotypes and tested in Uganda, Kenya, and Ethiopia, and they proved their resistance to stem rust disease, especially for this race [24]. Then, these two varieties were imported for cultivation in these countries to overcome this race (Ug99). As a result, most countries are attempting to speak one language in the field of wheat stem rust disease. As a result, it was critical to use a molecular marker to analyze the geographical connection and genetic diversity of newly generated races of the stem rust pathogen, P. graminis f. sp. tritici, across Africa, Asia, and Europe, as well as the Ug99 race.
Previously, Hasan and Abou-Zeid [25] reported that lines Sr31, Sr24, Sr38, and Sr36 were the most resistant genes, and Abebe et al. [26] reported that lines Sr24 and SrTmp were effective for all tested isolates; however, our findings do not support this point of view. Taken together, we suggest that changes in the behavior of some lines, i.e., Sr24, Sr31, Sr36, and Sr38, are responsible for resistance and had to be interpreted, especially with the first identification of the Ug99 [15].
As summarized by Singh et al. [12], Ug99 had been confirmed in Uganda (1998) Tanzania (2009). This pathotype/race can overcome many Sr genes in which virulence had not previously been detected. For instance, race Ug99 is the first known race of P. graminis f. sp. tritici that carries virulence to gene Sr31 located on 1BL. 1RS translocation from rye (Secale cereale) [27] as well as virulent to the majority of Sr resistance genes of wheat origin such as gene Sr38, introduced into wheat from Triticum ventricosum [20]. This strong virulence combination in Ug99 explains why wheat cultivars and germplasm are susceptible all across the world. Because of the breakdown of these critical genes, it is necessary to better understand the geographical association and genetic diversity of newly formed races within the Ug99 lineage of the stem rust pathogen in various wheat-producing locations in general, and Egypt in particular.
Fourteen stem rust races have been identified in Egypt and other countries in the stem rust greenhouse in Egypt and GRRC in Denmark. The geographical distribution of the most virulent races: TTKTT, TTKST, and TTKTK are found in Iran, Kenya, Rwanda, Tanzania, and Uganda), TTTSK is only found in Egypt, and TTKSK is found in Egypt, Iran, Kenya, Rwanda, Tanzania, and Uganda). The HCA and PCA-associated loading verified the substantial correlation between Kenya, Tanzania, Uganda, Rwanda, and Iran. Egypt, on the other hand, was adversely connected with the majority of the analyzed locations, including Eritrea, Spain, Ethiopia, Morocco, Italy, Poland, Kenya, Tanzania, and Uganda, or had no significant relationship, including Tunisia, Denmark, France, and Rwanda. Through this, it became clear that there is a relationship between the most virulent races (TTKSK, TTKTK, TTTSK, TTKST, TTKTT, TTRTF, and TTTTF) and the resistant monogenic lines (Sr11, Sr31, and Sr24) that fell into the same cluster through HCA and the PCA-associated loading.
Based on the North American naming system [28], isolates of Ug99 obtained in Kenya were identified as TTKS and were renamed TTKSK after adding a fifth set of differentials to the nomenclature system [20]. Given the gravity of this race, TTKSK (Ug99), it was vital to prove its existence using molecular markers in the majority of the nations where it was defined. The diagnostic PCR fragments indicate virulent TTKSK (Ug99) to Sr31, Clement (Sr31), and Brigardier (Sr31) as a fragment of 350 bp. This was in agreement with what was defined in the greenhouse (TTKSK) in Egypt and other countries based on the North American nomenclature [7,24].
Collectively, the findings of this study highlight the emergence of more aggressive races in Egypt, such as TTKSK, that break the resistance of wheat lines carrying Sr31, Sr24, Sr36, and Sr38 resistance. These aggressive races also appeared in some other countries such as Iran, Kenya, Rwanda, Tanzania, and Uganda. The TTTKS race is discovered for the first time in Egypt. As a result, the findings caution against breaking the resistance of Egyptian and international types bearing these genes. Because they were prone to infection with this disease, particularly the fungal races, it is vital in breeding efforts for rusts, particularly stem rust, to look for more efficient resistance genes and to incorporate more than one gene in one variety.
More research is needed to determine the genetic basis of existing wheat stem rust resistance in Egyptian wheat breeding materials (genotypes) and other nations. It is critical to understand the stem rust resistance genes that are now deployed in wheat cultivars in order to establish which genes provide long-lasting protection and more durable resistance in these wheat cultivars. This understanding will help alert pathologists and breeders to wheat cultivars' genetic vulnerability in addition to assisting us in developing diseaseresistant varieties and how to trade these types with one another in order to ensure global food security.

Conclusions
Due to their migration to countries with the bulk of the stem rust resistance genes (Sr) and the complex virulence combinations they carry, the Ug99 group of wheat stem rust fungus races continue to pose a threat to the global supply of wheat. In Central West Asia (CWANA), 10 African nations, 10 European nations, and at least 14 stem rust races have been recognized. Four races-TTKSK, TTKST, TTKTK, and TTKTT-came from five African nations-Egypt, Kenya, Rwanda, Tanzania, and Uganda-as well as Iran-but none from Europe-in the Ug99 group. On the other hand, TTRTF, TKTTF, and TTTTF were not discovered in Egypt. Kenya, Tanzania, Uganda, Rwanda, and Iran were all closely associated with one another, according to a correlation analysis. Contrarily, Egypt had a negative correlation with the majority of the countries examined, including Eritrea, Spain, Ethiopia, Morocco, Italy, Poland, Kenya, Tanzania, and Uganda, or at least no significant correlation was found, including Tunisia, Denmark, France, and Rwanda. By using molecular markers, Ug99 (TTKSK) was validated in Egypt. Only in Egypt did the TTTSK race become known.