What Was the Reason for the Durable Effect of Sr31 against Wheat Stem Rust?

Abstract

Common wheat cultivars have been protected from stem rust for several decades worldwide by the Sr31 resistance gene transferred from Secale cereale L. (cv. Petkus). In 1998, Sr31 was overcome in Uganda by the Ug99 race of Puccinia graminis f. sp. tritici Eriks. & Henn. (Pgt). The Ug99 race and its derivatives have spread widely in Africa, neighboring regions and Europe. However, Sr31 remains effective in other areas of the world, including Russia. To breed wheat with durable resistance, it is promising to research the resistance mechanisms of nonhost species and introgressive cultivars. The aim of the research was to estimate the resistance of S. cereale and Triticum aestivum cultivars with Sr31 to stem rust and to study the mechanisms of incompatibility of Pgt with plants at the cellular level. The research was carried out in Western Siberia (Russia, Omsk region) in 2018–2022. Rye and wheat with Sr31 (cvs. Kavkaz, Seri 82, Bacanora (=Kauz’s’), NIL Thatcher TcLr26/Sr31) were resistant at the stages of seedling and adult plant, and cv. PWB343 was more susceptible to disease. Cytological studies have shown that Pgt died on the rye plants on the surface, and cv. Petkus intensively suppressed the development of the appressoria necessary to penetrate into tissues. Wheat cultivars inhibited the Pgt development mainly on the surface and while it attempted to penetrate into the stomata (pre-haustorial resistance). It has been demonstrated that Pgt has to adapt step-by-step to the surface and tissue properties for compatible interaction, which may be the reason for the durable effectiveness of Sr31.

1. Introduction

Common wheat (Triticum aestivum L.) is one of the most important cereals. Its crops occupy more than 200 million hectares worldwide. To provide food for the growing population of the planet, it is necessary to increase the production of grains by 70% by 2050 [1]. To increase the grain harvests, it is useful to increase the efficiency of photosynthesis and the potential productivity of plants, as well to reduce losses from biotic stress factors, including fungal diseases. Most significant damages in wheat production are caused by stem, leaf, and stripe rusts which are caused by the biotrophic fungi (Puccinia graminis f. sp. tritici Eriks. & Henn., P. triticina Eriks. and P. striiformis Westend f. sp. tritici Eriks., respectively) [2]. In the first half of the twentieth century, stem rust was considered the most serious threat in North and South America, Australia, Europe, Africa and Southeast Asia [3,4,5,6,7]. Due to its harmfulness, stem rust was the first object of scientific immunological and genetic research. At the beginning of the twentieth century, specialized forms (formae specialis—f. sp.) to main cereals (wheat, rye, barley, etc.) were revealed within the species P. graminis, and within them, the physiological races were determined [8,9]. After the identification of resistance genes, cultivars resistant to the disease were breed. A positive effect on the decrease in stem rust was exerted by the removal in different regions of an alternative host—Berberis spp., on which P. graminis f. sp. tritici (Pgt) passes the sexual stage, which leads to recombinations and increases the polymorphism of its populations [10].

Using breeding cultivars defended by resistance genes, including alien ones, is a generally recognized way to protect crops from diseases. The translocation 1BL/1RS with a complex of resistance genes to leaf, stem and stripe rusts, Lr26/Sr31/Yr9, was introgressed to wheat from the winter rye cv. Petkus in Germany in the 1930s [11]. Cultivars Aurora, Kavkaz and Lovrin, having this translocation, have been widely used as parent forms in wheat breeding worldwide [12]. The spread of cultivars protected by the Sr31 led to the suppression of Pgt reproduction and made stem rust economically insignificant for several decades [13]. However, in Uganda in 1998, the Ug99 race (TTKSK) appeared which overcame Sr31, and over time, 13 races with new combinations of virulence genes (Ug99 group) emerged within it [6,14]. An additional increase in stem rust development in Africa and Europe was associated with the appearance of avirulent to Sr31 races (Digalu and Sicilian) unrelated to the Ug99 group [15]. Within two decades, stem rust spread throughout Africa, the Middle East, and most European countries, including northern Great Britain, Ireland and Sweden [6,15,16]. In Russia, common wheat crops occupy millions of hectares in the European and Asian parts of the country. In the second half of the twentieth century, stem rust developed occasionally in the European part, mainly in the North Caucasus and the Volga region. In 2010 and 2013, there was an increase in stem rust development in the Central Black Earth and the Volga regions, and in 2016 an epidemic was recorded in the Volga region [17,18]. In Western Siberia and Northern Kazakhstan, the first serious stem rust epidemic in recent decades occurred in 2015 in the “wheat belt,” which occupies several millions of hectares [19]. The research of Pgt populations existing in European countries in terms of virulence and molecular markers showed that they consist of clone groups (clades), both belonging to the Ug99 group and independent of it [20,21]. Russian Pgt populations differ from European ones and represent a separate clade. There is a separate Asian Pgt population in the territory of Western Siberia, in which two subpopulations (Omsk and Altai) have been identified [22]. The Omsk subpopulation has the most pathotype polymorphism and is avirulent to Sr31, and the Altai one is avirulent to Sr24, Sr30 and Sr31. The polymorphism of the Omsk Pgt population is presumably associated with the presence of Berberis spp. plants in the region [23].

Previously, it was noted that races with virulence toward Sr24, Sr31 (with the exception of the Ug99 group spreading zone) and Sr38 are rarely found in the world [24]. Due to the sharp increase in stem rust, the phytopathological situation in the grain regions worldwide is under close control. In population samples collected in the Volga region (Saratov) in 2013–2014 and in Western Siberia (Omsk) in 2016, single isolates virulent toward Sr31 were identified [17,21], but the Ug99 race has not been determined in Russia [21,22]. Significant accumulation of virulent clones in the following years were not determined, and Sr31 remains effective in Russian regions [25,26]. In addition, Sr31 was effective in other regions, including the USA and Canada, India, China, and the Republic of Kazakhstan [27,28,29,30,31].

Experience in crop production has shown that due to the high variability of rust fungi, the resistance of most cultivars (with rare exceptions) have been quickly overcome [27,32]. In 1983, R. Johnson formulated the concept of “durable resistance” as resistance remaining effective in a widely cultivated cultivar for a long period, under environmental conditions favorable for disease development [33]. However, as the famous phytopathologist J.E. Parlevliet noted, the durability of resistance is a function of time, and it is overcome sooner or later [34]. The situation with long-period suppression of stem rust worldwide (up to the appearance of Ug99 race) indicates that Sr31 provided durable cultivar resistance. However, the action of the Sr31 gene has not been studied before.

To understand the mechanisms of durable resistance, it is important to study interactions between pathogens and species to which they are not adapted/specialized—nonhost species. The research of nonhost resistance (NHR) is considered a strategic direction for breeding cultivars with durable resistance to diseases [35,36]. Pgt does not damage S. cereale, so the rye is a nonhost species for it. Due to the fact that the Omsk Pgt subpopulation is aggressive and polymorphic, the study of the interactions between pathogen and S. cereale and T. aestivum cultivars is promising to understand NHR and the reasons for the durable effect of the Sr31 gene.

Pgt is one of the highly specialized biotrophic rust pathogens. The fungus progressively forms a set of infection structures to interact with plant. After the attachment of the spores to the surface, growing tubes are formed, directed to the stomata. At the ends of the growing tubes, the appressoria are developed, which provide penetration into the stomatal opening and formation of substomal vesicles in the substomal cavity. Then, colonies with branched infection hyphae are developed in the tissues. Rust fungi form specialized intracellular structures—haustorium for uptake of nutrients from the plant [37]. In compatible interaction, pustules with the next generation of urediniospores are developed. Previously, it was found that in nonhosts, the development of non-specialized fungi is stopped at the early stages of development [38,39]. Cultivar resistance appears after overcoming NHR, and as a rule, is accompanied by a hypersensitivity reaction (HR) [36,40]. In the 1960s–1970s, a complex of cytological studies of interactions between Pgt and resistant wheat cultivars and lines with race-specific Sr5, Sr6, Sr8, and Sr22 was carried out [41,42,43,44,45]. The most attention was paid to the details of HR and its influence on the fungus’s development. After the elimination of the stem rust threat, investigations of resistance mechanisms to stem rust were stopped for a long period. Later, the effects of the Sr35 and Sr50 genes were studied using various methods [46,47].

The aim of the research was to estimate the resistance of S. cereale samples and T. aestivum cultivars with Sr31 to the Omsk Pgt population and to study the NHR mechanisms and effect of Sr31 in plants at the cellular level.

2. Materials and Methods

2.1. Plant Material

The experimental materials included:

• Spring common wheat T. aestivum with the identified Sr31—cvs. Kavkaz, Seri 82, Bacanora (=Kauz’s’), PWB343 and a near-isogenic line of cv. Thatcher (RL-6078, NIL-THATCHER-Lr26,Sr31-ST-1-25[000]) (TcLr26/Sr31).
• Spring common wheat that is susceptible to stem rust—standard cvs. Pamyati Azieva (medium-early) and Duet (medium-ripe); wheat that is universally susceptible to leaf diseases —cv. Chernyava 13.
• Winter rye, S. cereale—cvs. Petkus, Siberia, Irina, Vavilovskayja universalnaya, Zilant, Tyumenka, Evrika, line 22/18 (originated in the Omsk Agrarian Scientific Center—Omsk ASC, from combination Jubileynayja 25 × Tetra short). According to the information provided in the Plant Genetic Resources Database of Federal Research Center N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR, St. Petersburg, Russia) cv. Petkus, it is present in the pedigrees of rye cultivars and line 22/18 (http://db.vir.nw.ru/virdb (accessed on 1 September 2022))

Seeds of cvs. Kavkaz (spring, k-52721) and Petkus (tetra, k-10254) were obtained from the National Genebank of the Russian Federation (VIR), the others were provided by the Omsk ASC. The samples were multiplied and maintained on the basis of the Laboratory of field crops breeding of the Omsk State Agrarian University (Omsk SAU).

2.2. Infection Material

The development of stem rust in the field was studied on the natural infection background in the South of Western Siberia (Omsk, 54.58_ N, 73.24_ E). In 2018–2020, the disease developed on crops annually. The first symptoms were observed in the second and third ten days of July, and the maximum severity in the second and third ten days of August.

For laboratory studies, damaged stems of commercial cultivars and breeding lines of spring common wheat were collected in the experimental fields of the Omsk SAU in 2020. Samples of Pgt urediniospores were revitalized and propagated on susceptible plants of cv. Pamyati Azieva. To infect a set of rye samples, the population inoculum propagated on susceptible wheat was used. The same population inoculum, and monopuctule isolates from it, were used to infect the resistant wheat.

2.3. Estimation of Stem Rust Development in the Field and Laboratory Conditions

Field experiments were carried out in the forest-steppe zone of Western Siberia in 2017–2020. Winter rye cultivars were sown in the third ten days of August 2017–2019, and spring cultivars in the third ten days of May accordingly. The samples were sown on plots 1 m², with a seeding density of 500 grains/m². The development of the disease on wheat was estimated on adult plants in dynamics from the second ten days of July to the third ten days of August. The final assessment was performed at the beginning of wax maturity (Zadoks, ph. 82). The rust development on winter rye was estimated twice—in the tillering phase (September) and before harvesting (second half of July). The stem rust symptoms were determined according to the CIMMYT methodology. Infection type was estimated according to the Roelfs scale [48] and disease severity on a 0–100% modified Peterson scale [49].

In the laboratory, the development of stem rust was studied on 10-day-old seedlings grown in pots. Plants were infected by shaking spores onto the moistened surfaces of the plants. Infected plants were cultivated the first day at a temperature of 26–27 °C, and later at 23–25 °C under illumination with an intensity of 10,000 lux for 16 h. Infection types (ITs) were determined by a modified Stackman scale [50]: (0—without symptoms;;—necrotic flecks without pustules—immunity; 1—necrotic flecks and small pustules surrounded by necrotic zones; 1–2 small pustules surrounded by necrotic zones of various sizes; 3+−large pustules surrounded by chlorotic zones; 4—large pustules). ITs 0–2 were classified as resistant, and 3–4—as susceptible. For the control in laboratory studies, the susceptible wheat cv. Pamyati Azieva was used.

2.4. Cytological Studies

The leaves infected with Pgt were used for cytological studies. The fixation of the material was carried out 1, 2, 3, 5, and 10 days after inoculation in a lactofenol mixture (lactic acid:phenol:glycerin:water:96% ethanol = 1:1:1:1:8) by boiling until the leaves discolored. Differential staining of pathogen structures was performed using the modified Shipton and Brown method [51]. The leaves were stained with 1% aniline blue in lactophenol at 60 °C for 0.5 h; then, the color was differentiated in a saturated chloral hydrate solution (2.5 g chloral hydrate/mL H₂O) for 2–3 h at 60 °C. As a result, the intact fungus structures were stained light blue; the damaged ones, dark blue; the intact plant cells, light blue; and the cells that died as a result of HR, dark blue. The studies were carried out using light microscope Micromed-5 (LOMO, Russia).

To study the interactions of Pgt with cultivars of the cross-pollinated species S. secale, 8 plants per variant were used, along with the self-pollinated species T. aestivum—4 plants per variant. The results of Pgt interaction with each plant were considered as a repetition of the variant. The results of the development of 20–30 spores per plant were determined. To characterize the Pgt—plant interactions, some indicators were determined: the numbers of growing and non-growing spores, the number of growing tubes with appressoria, the number of appressoria on stomata and on the surface, the number of substomal vesicles, the number of colonies per plant.

To identify the features of the Pgt interactions between wheat and rye samples, some indicators were calculated:

• − The proportion of germinated spores with growing tubes of the total number of spores (germinated and non-germinated) on the plant (in %);
• − The proportion of growing tubes developed appressoria of the total number of germinated spores with growing tubes (in %);
• − The proportion of appressoria on stomata from their total number on plant surface (in %);
• − The proportion of appressoria germinated into stomata (formed substomal vesicles) from the total number on the stomata (%).

The diameters of the colonies (20 ones) were measured in the 10 days after inoculation. The mean values of cytological data and the error of the mean (M ± SEM) were calculated.

3. Results

3.1. Estimation of Stem Rust Development in the Field and Laboratory

Weather conditions favored the development of stem rust on crops during the research period. The maximum damage of susceptible cultivars was noted in 2018 and 2019 (70MS-100S), and lesser damage in 2020 (20-50S). The cvs. Pamyati Azieva and Chernyava 13 were hit to the maximum extent (

Table 1. Results of estimation of stem rust development on cultivars and lines of T. aestivum. and S. cereale in the field (Western Siberia, Omsk) and in the laboratory.

Cultivar, Line	Field, IT & Severity			Laboratory, IT *
	2018	2019	2020
Triticumaestivum
Pamyati Azieva	100S	100S	50S	4
Duet	80S	70MS	20S	4
Chernyava 13	100S	100S	50S	4
TcLr26/Sr31	5M	0	0	0
Kavkaz	10MR	0	0	0
Seri 82	5MR	0	0	0
Bacanora	5M	0	0	;1
PWB343	30MS	20MS	10MS	;, 2−, 3+
Secale sereale
Petkus	0	0	-	0
Siberia	0	0	-	0
Irina	0	5MR	-	;
Line 22/18	0	0	-	;
Vavilovskayja universalnaya	0	0	-	0
Tyumenka	0	0	-	0
Zilant	0	0	-	;
Evrika	0	0	-	0

Note: * infection with urediniospores of Pgt population (2020), propagated on a susceptible wheat cv. Pamyati Azieva.

). Stem rust developed on cvs. Kavkaz, Seri 82, Bacanora and TcLr26/Sr31 to a weak degree (5-10MR-M) in 2018, in the following years, these cultivars had no disease symptoms. Cv. PWB343 showed moderate severity and susceptible infection type (30MS) in 2018, and later the severity was decreased, although the susceptible infection type remained (10S). Most rye cultivars showed immunity to the disease at the tillering (September 2017–2019) and the ripening stages (July 2018–2019). In 2019, single small pustules surrounded by necrotic zones (5MR) were found on individual stems of the cv. Irina before harvesting. The infected stems were collected, but the attempts to revitalize the fungus were unsuccessful. In 2020, rye ripened before the appearance of stem rust, so the estimation was not performed.

The seedling resistance test was carried out on wheat and rye samples when infected with Pgt population inoculum. Cultivars Pamyaty Azieva, Duet and Chernyava 13 confirmed the susceptibility noted in the field (IT 4) (Table 1, Figure 1a). There were no signs of infection on cvs. Kavkaz and Seri 82 and line TcLr26/Sr31 (IT 0, immunity). On the cv. Bacanora appeared small necrotic flecks and single small pustules surrounded by necrotic zones (IT; 1). On cv. PWB343, necrotic flecks and pustules of various sizes with necrosis or chlorosis were noted (IT;, 2−, 3+) (Figure 1b–f). There were no pustules on rye, but necrotic zones of different sizes appeared on cvs. Irina, Zilant and line 22/18 (IT ;) (Figure 1g–n).

Additionally, the reaction of wheat cultivars to infection with 14 Pgt isolates was estimated. Cvs. Kavkaz and Seri 82 and line TcLr26/Sr31 were immune to all isolates, but minor necrosis appeared in some combinations (IT 0, ;) (

Table 2. Results of laboratory evaluation of T. aestivum seedlings’ reactions to infection of. P. graminis f. sp. tritici isolates, infection type.

Cultivarline Isolate	Pamyati Azieva—Control	TcLr26/Sr31	Kavkaz	Seri 82	Bacanora	PWB343
1	4	0	0	0	0	0
2	4	0	0	;	;	3+
3	4	0	0	0	0	0
4	4	0	0	;	;	0
5	4	0	0	0	0	0
6	4	0	0	0	0	0
7	4	0	0	0	;	1
8	3	0	0	;	0	0
9	4	0	0	;	0	0
10	3	0	0	0	;1	2
11	4	0	0	;	;1	2
12	3	0	0	0	1, 2	3+
13	4	;	;	0	;, 2, 3+	3+
14	4	0	0	;	0	0

). Cv. Bacanora showed immunity to 10 isolates (IT 0, ;) and resistance to three (IT 1, 2); and when infected with isolate 13, pustules with different sizes and necrosis or chlorosis appeared (IT ;, 2, 3+). Cv. PWB343 was immune to eight isolates, resistant to three and susceptible to three (IT 3+). When infected with isolates 11, 12 and 13, the reactions of cv. PWB343 were shifted towards greater compatibility compared to cv. Bacanora.

3.2. Results of Cytological Research of Pgt Interactions with Rye and Wheat

Cytological studies have shown that on the surface of susceptible cv. Pamyati Azieva, the spores attached to the surface and 93% of them germinated, forming growing tubes. The growing tubes moved to the stomata, and most of them (85%) formed appressoria, mainly on the stomata (88%) (Figure 2a;

Table 3. Results of the development of infection structures of P. graminis f. sp. tritici on the surfaces of rye plants *.

Caltivar, Line	Violation of Spore Adhesion **	Proportion of Germinated Spores, %	Proportion of Growing Tubes with Appressoria, %	Proportion of Appressoria, %
				On Stomata from the Total Number	Germinated from the Number on the Stomata
Pamyati Azieva—control	-	93.1 ± 3.9	85.1 ± 2.7	88.0 ± 3.2	83.2 ± 1.0
Petkus	+	62.7 ± 1.3	17.8 ± 1.2	43.8 ± 2.1	29.1 ± 2.1
Siberia	++	46.9 ± 1.2	59.3 ± 2.4	24.2 ± 2.0	38.4 ± 2.4
Irina	+	57.4 ± 1.2	25.0 ± 1.3	34.5 ± 1.9	12.5 ± 1.7
Line 22/18	-	61.2 ± 1.3	34.2 ± 1.9	25.7 ± 1.9	26.6 ± 2.4
Vavilovskayja universalnaya	+	62.5 ± 1.4	31.4 ± 1.6	18.2 ± 1.3	30.6 ± 2.1
Tyumenka	++	46.3 ± 1.3	61.5 ± 2.0	30.1 ± 1.8	43.7 ± 2.5
Zilant	+	59.1 ± 1.2	49.6 ± 1.8	34.6 ± 1.7	29.3 ± 2.1
Evrika	-	65.8 ± 1.5	32.8 ± 1.7	45.1 ± 2.1	27.2 ± 2.1

Note: * infection with urediniospores of the Pgt population propagated on a susceptible cv. Pamyati Azieva; ** “-”—absent, “+”—weak. “++” moderate.

). Then, the cytoplasm of appressoria relocated into the substomal vesicles in the substomal cavity, and empty shells remained on the guard cells. Later, the infection hyphae entered from the substomal vesicles, at the ends of which, the specialized haustorial mother cells were formed (HMC). HMC were attached to the cellular walls and formed haustorium in mesophyll cells (Figure 2b). In the tissues of the susceptible cultivar, the colonies progressively developed and formed the pustules with the next generation of urediniospores (Figure 2c). When studying infected rye plants, it was found that parts of the spores (died and germinated) moved under the cover glasses, which indicates a violation of spore adhesion to the surface. This feature was most pronounced in the cvs. Siberia and Tyumenka (25–30% of spores moved), and to a lesser extent—in the cvs. Petkus, Irina, Vavilovskaya universalnaya and Zilant (10–15%). On all rye samples, spore germination was significantly suppressed (by 1.5–2 times compared to the control), which was especially evident on cvs. Siberia and Tyumenka, which showed depressed spore adhesion (Table 3). The rye cultivars significantly suppressed the appressoria formation, which was most pronounced on cv. Petkus (4.8 times compared to the control), and on cvs. Irina, Vavilovskaya universalnaya and Evrika and line 22/18 (2.6–3.5 times). In addition, on all rye, a smaller amount of the appressoria (18–44%) were located on the stomata, and the rest, on the epidermal cells (Figure 2d). This indicates the violation of germinating tubes’ orientation to the stomata. This was most pronounced on cvs. Vavilovskaya universalnaya and Siberia and the line 22/18. In rye, a small part of appressoria (12–44%) formed a peg into the stomatal slit and stopped at the stage of substomal vesicle, showing no attempts to penetrate into the cells (Figure 2f). In other cases, the cytoplasm remained in the appressorium and showed no signs of degradation for 2 days. Often, the substomal vesicle and infection hypha were located on the surfaces of plants, but not in the substomal cavity (Figure 2g). The appearance of small necrosis on cvs. Irina and Zilant and line 22/18 was associated with the plant’s reactions to the dead surface fungus structures (spores and appressoria) at the late stages of the experiment, but not the manifestation of HR.

The effect of introgressed Sr31 in various wheat genotypes was studied on the examples of interactions with four Pgt isolates (

Table 4. Results of the development of infection structures of P. graminis f. sp. tritici on the surfaces of common wheat samples with Sr31.

Cultivar, Line	Isolate	IT	Proportion of Germinated Spores, %	Proportion of Growing Tubes with Appressoria, %	Proportion of Appressoria, %
					On Stomata of the Total Number	Germinated from the Number on the Stomata
Pamyati Azieva—control	1	4	93.0 ± 3.9	85.0 ± 2.7	88.0 ± 3.2	83.0 ± 1.0
	10	3+	88.9 ± 3.5	83.1 ± 2.9	82.5 ± 3.5	89.1 ± 2.3
	11	4	92.1 ± 2.6	80.9 ± 3.1	85.1 ± 1.9	92.3 ± 1.8
	13	4	88.5 ± 2.3	87.9 ± 2.5	90.2 ± 2.3	94.3 ± 2.1
TcLr26/Sr31	1	0	84.2 ± 1.2	45.9 ± 1.9	65.3 ± 1.6	8.9 ± 1.1
	10	0	88.3 ± 1.6	56.9 ± 1.4	61.8 ± 1.4	12.1 ± 1.5
	11	0	86.5 ± 1.3	60.4 ± 1.2	71.4 ± 1.2	13.3 ± 1.4
Seri 82	1	0	86.4 ± 1.0	41.5 ± 1.2	76.2 ± 0.9	10.3 ± 1.3
	10	0	83.5 ± 1.7	65.2 ± 2.8	67.8 ± 2.8	31.2 ± 2.8
	11	;	88.6 ± 0.7	78.4 ± 1.8	85.1 ± 0.9	7.5 ± 1.0
Bacanora	1	0	87.3 ± 1.2	56.1 ± 1.7	68.2 ± 1.4	33.1 ± 1.5
	10	0	88.8 ± 1.2	60.3 ± 1.6	85.0 ± 0.9	7.6 ± 0.9
	11	;1	91.8 ± 1.2	33.4 ± 2.1	65.5 ± 2.8	23.3 ± 2.9
	13	;, 2, 3+	84.0 ± 1.5	82.4 ± 2.1	91.7 ± 1.2	35.4 ± 1.3
PWB343	1	0	89.1 ± 1.3	68.9 ± 1.7	72.4 ± 1.1	41.2 ± 1.4
	10	2	98.2 ± 2.1	79.3 ± 1.6	84.6 ± 1.8	59.4 ± 2.5
	11	3+	94.5 ± 2.5	83.4 ± 2.1	77.6 ± 1.3	66.4 ± 2.1

). The choice of isolates was determined by the fact that the cultivars showed the same IT as the Pgt population. Cytological studies have not revealed a violation of spore attachment, such as big differences in its germination on wheat. On the surface of resistant wheat cultivars, the orientation of the growing tubes was improved compared to rye, although in 9 of the 13 studied variants it was lower than in the control (61.8–76.9%). In most combinations, the appressorium development was increased compared to the cv. Petkus (18%), but was significantly suppressed compared to the control (by 1.3–2.5 times). New appearance of resistance to Pgt isolates was a sharp inhibition of penetration into the stomata on cvs. Seri 82 and Bacanora and TcLr26/Sr31 (7.6–23.3%, as opposed to 29.1% on cv. Petkus). At the same time, the cytoplasm remained in the appressorium and was intensively stained, which indicates its destruction. In rare cases, a substomal vesicle and primary infection hyphae were formed (Figure 2h,i). Cultivars Seri 82 and Bacanora and the line TcLr26/Sr31 had the greatest negative impact on the development of Pgt surface structures. When isolates 1, 10 and 11 interacted with cvs. Seri 82 and Bacanora and the TcLr26/Sr31 line, it was shown that important properties for penetration into plants changed independently (orientation to stomata, intensity of appressorium development, and ability to penetrate into stomata). For the cultivar most susceptible in the field and laboratory, cv. PWB343, all the isolates developed more successfully than on other samples with Sr31.

On cvs. Bacanora and PWB343, some isolates were able to grow the next generation of urediniospores, which indicates that they overcame part of the resistance mechanisms. To study the relationships in overcoming the resistance of the cv. Bacanora, isolate 13 (IT ;, 2, 3) was used. The development of this isolate in the orientation of the growing tubes toward the stomata and the intensity of the formation of appressoria was similar to the control, but penetration into the stomata was suppressed (35% of germinated appressoria versus 80–85% in the control) (Table 4). After penetration into the stomata, in 60% of infection sites, the development was stopped at the stage of a substomal vesicle or single infection hyphae (similar to Figure 2f,i). Colonies of different sizes were formed in the remaining infection sites (Figure 3). In the smallest colonies, the fungus stopped development after the formation of 2–3 hyphae and 1–2 haustorium in plant cells. The single plant cells died after haustoria penetration as a result of HR (Figure 2j). Later, the necrotic zones expanded due to the destruction of cells adjacent to the dead colonies. In some infection sites, small colonies (101–300 µm in diameter) were aborted without HR, and their hyphae eventually vacuolized and died (Figure 2k). In some of infection sites (8.5%), despite the occurrence of the HR, larger colonies developed (up to 600 µm), as did medium-sized pustules (IT 2). In rare cases, large colonies were formed, and pustules surrounded by chlorotic zones were formed (Figure 2l). Such results indicate that the isolate acquired properties that allow it to develop successfully on the surface, but it unstably interacted with plant tissues.

4. Discussion

Due to the high ecological plasticity, common wheat is cultivated in the most agricultural zones of the planet. The giant populations of rust fungi exist on crops, in which evolutionary processes are constantly taking place, aim to overcome the cultivar’s resistance. Leaf rust regularly affects crops, but in recent decades, wheat has been strongly struck by stem and stripe rusts in different regions of the world [21,27,52,53]. One of explanations for the accelerated evolution of pathogens is climate change. With warming and an increase in the CO₂ content in the atmosphere, plants form a large biomass and leaf canopy, which contributes to the reproduction of biotrophic fungi. At the same time, the probabilities of mutations in virulence genes and modifiers affecting the strain fitness increase. Additional risks are created by the migration of pathogens, which increases due to the instability of atmospheric processes and the transfer of cyclonic masses along new trajectories [54]. The organization of large-scale monocropping promotes the selection and survival of new races in agrocenoses. In this regard, the research of durable resistance mechanisms to diseases is especially relevant.

The studies were carried out in the Omsk region, where wheat crops occupy more than 1.4 million ha, and rye, about 4 thousand ha (https://omsk.gks.ru/storage/mediabank (accessed on 1 September 2022)). Significant damage of wheat crops by stem rust, up to the epidemic of 2015, was not observed for several decades. In 2016–2017, disease development was strong but did not reach the epidemic level [19]. According to our data, in 2018 and 2019, stem rust intensively affected wheat before harvesting (up to 100S), and in 2020–2022 its development was limited by drought. During these years, there were not symptoms of stem rust on commercial and breeding rye crops (Yu. N. Kashuba, unpublished).

Earlier, it was shown in the Spanish regions that near the plantings of the alternative host Berberis spp., Pgt polymorphism increased sharply, and even cross-infection with forms of P. graminis existed on wheat, rye and wild grasses [21]. Our experiments with rye were carried out on a winter wheat field, in the immediate vicinity of Berberis bushes (about 500 m). Most of the rye samples were immune to stem rust during the research period. The exception was cv. Irina, on which a small number of Pgt pustules (5 MR) were found in 2019. It is unclear whether the symptoms re associated with the development of a wheat or rye formae specialis, since the infection was not revitalized. Taking into account the fact that there are Berberis spp. plantings in the Omsk region, the phytopathological situation will be monitored in crops in the future.

Observations of the development of stem rust on cultivars with Sr31 showed periodic changes in their resistance in the Republic of Kazakhstan and Western Siberia. In 2006–2007, cvs. Seri 82 and PBW343 were damaged (20–30 MS) in Kazakhstan [55]. In the Omsk region in epidemics of 2015, the cv. PWB343 was damaged up to 80 S, but disease declined sharply in 2016 (10 R). At the same time, cvs. Seri 82 and Bacanora were resistant, which demonstrates the different Sr31 effects in the cultivars [56]. Our observations have confirmed the high resistance of cvs. Kavkaz, Seri 82 and Bacanora and the line TcLr26/Sr31 in 2018–2020; and cv. PWB343 was affected to a moderate or low degree. These facts confirm the proposal that virulent mutations to Sr31 gene can occur in different Pgt populations, regardless of the Ug99 race [21]. Probably, virulent clones had low fitness in Western Siberia and the Republic of Kazakhstan, since the damage of cultivars defended by the Sr31 gene decreased, and the elimination of virulent clones from Pgt populations was noted [25,49]. However, under favorable conditions and with a wide spreading of cultivars, there is a high probability to overcome resistance, which happened with the genes in the 1BL/1RS translocation. The Lr26 gene was overcome in southern Russia and Ukraine in 1973 after sowing the cvs. Kavkaz and Aurora on more than 5 million ha [57], and the Lr26 and Yr9 genes were overcome in India in 1983–1986 when cultivating the cv. PWB343 on the territory of 7 million ha [58].

Due to the need for durable protection of crops from rapidly progressing diseases, interest in the study of NHR has increased. Long-term observations have shown that NHR is stable; overcoming it occurs extremely rarely due to the expansion of the range of pathogen hosts (jamp) [59,60]. In the 1970s, a general concept of the interaction between pathogens and plants was formulated on the basis of basic compatibility and resistance. It was assumed that in the course of evolution, specialized species and forms should acquire properties allowing them to exist on plants, and to overcome the mechanisms of basic resistance; after that, the species becomes the “host” [61,62]. In the 1980–1990s, it was shown that there is a temporal signal exchange between partners during pathogenesis [63].

Later, a two-level model of active plant defense was proposed, based on the existence of two types of resistance—nonhost and host (cultivar). It was assumed that upon contact with microorganisms, the surface molecular receptors of plants PRRs (pattern recognition receptors) recognize a set of conserved molecules within the species: MAMPs (microbe-associated molecular patterns), PAMPs (pathogen-associated molecular patterns) and DAMPs (damage-associated molecular patterns, products of destruction of plant cells). As a result of recognition, the first level of defense of nonhosts is triggered—PAMP-triggered immunity (PTI). To overcome the basic resistance of nonhosts, pathogens have to acquire a set of effectors corresponding to the species [64,65]. After overcoming PTI, the second level of protection is activated, associated with the recognition of specific effectors—ETI (effector-triggered immunity). Host receptor NB-LRR proteins encoded by “major” resistance genes recognize the race-specific pathogen effectors and trigger a signaling cascade to implement defense reactions. The action of ETI corresponds to the cultivar resistance described by the theory of H. Flor “gene-for-gene” and is usually accompanied by an oxidative burst and HR [65,66]. Over time, it became clear that the signaling pathways of PTI and ETI can overlap, especially in host-related species [66]. According to modern concepts, plants also possess preformed constitutional barriers (physical, chemical), which form an essential part of the basic resistance [35,67].

A complex of previously performed studies has shown that the incompatibility of rust fungi with nonhosts manifests itself at early stages, but the results depend on the pathosystems. When infecting species out of the Poaceae, the development of Pgt and P. triticina fungi stopped at the stage of growing tubes [37,68]. P. triticina practically did not form appressoria on the corn Zea mays, millet Panicum miliaceaum and rice Oryza sativa, and rare appressoria were not able to provide penetration into stomata [68,69]. The formation of P. triticina appressoria was twice suppressed on the surfaces of immune species Thinopyrum ponticum and Avena sativa, and all of them died on stomata as a result of ROS (superoxide O₂^•–) generation [68,70]. The barley rust fungus P. hordei did not form the appressoria on a resistant Hordeum chilense [71], but in other nonhosts stopped after penetration into stomata but before haustorial invasion into plant cells—i.e., so-called pre-haustorial resistance was manifested [38,72]. At the same time, P. graminis f. spp. secalis and avenae were able to form colonies of various sizes in rice that never rusted. The rust fungi Uromyces vignae and U. appendiculatus in Fabae species close to their hosts developed small colonies with HR [67,72]. Pre-haustorial resistance is recognized as the extreme incompatibility of rust fungi with nonhost species [38,39,72].

Our results showed that the development of Pgt on rye stopped on the surfaces of the plants, i.e., pre-haustorial resistance appeared. Incompatibility with rye was manifested at several stages: the spore attachment and germination, the detection of stomata by growing tubes, the formation of appressoria and penetration into the stomata. The cv. Petkus inhibited the Pgt development at all stages, but to the greatest extent suppressed the appressoria formation. Such results indicate the existence of common rye NHR mechanisms to Pgt and quantitative differences in their appearance between cultivars.

In the opinion of J. Kolmer [27], durable resistance of wheat cultivars is difficult to achieve, due to the high variability of rust fungi, and for this purpose, genotypes based on effective resistance genes should be created. To understand the basis of durable resistance, it is important to investigate the action of introgressed genes in the wheat cultivars. The Sr35 gene (from T. monococcum) inhibited Pgt development after penetration into the stomata at the stage of infection hypha [46]. The Lr19 gene (from Th. ponticum) provided an effect similar to the donor nonhost, i.e., a strong suppression of appressoria formation and their death on stomata after an oxidative burst [51]. A similar effect was exerted by the Lr9 gene (from Ae. umbellulata), and its action was not changed in different genetic backgrounds [73].

When researching the Sr31 gene’s action in wheat samples, it was found that some of the incompatibility factors were lost (weak adhesion and spore germination). However, when most isolates developed on cvs. Seri 82 and Bacanora and the line TcLr26/Sr31, growing tubes’ orientation toward the stomata and the formation of appressoria were suppressed, although the inhibition was weaker compared to rye, especially in cv. PWB343. Unlike rye, in cvs. Seri 82 and Bacanora and TcLr26/Sr31, an additional defense mechanism appeared in the form of destruction of appressoria and substomal vesicles, similar to Lr19′s action [51]. This indicates that the cv. Petkus’s features were partially transferred to wheat cultivars, but the action of alien genes in the 1BL/1RS translocation was enough to realize the pre-haustorial resistance to Pgt.

On the whole, these facts demonstrate that nonhosts, filogenically far from T. aestivum, may possess effective preformed preinvasion barriers against wheat rust fungi. The first PTI reaction in the form of ROS generation occurs after the appressoria has close contacts with stomatal guard cells. It is important that such defense mechanisms can be transferred monogenously or as part of a complex translocation to wheat cultivars.

To understand the coevolution processes occurring when overcoming the Sr31 gene, the interactions of cvs. Bacanora and PWB343 with a set of isolates with different IT were compared. It was found that, as IT changed from resistant to susceptible, the orientation to stomata, the appressoria’s development and penetration into the stomata were enhanced, and such properties changed independently. This indicates that to overcome the Sr31 gene, the pathogen has to undergo several adaptations to the plants’ features, and these microadaptations are not available for visual inspection. Previously, a similar multi-step adaptation of P. triticina to the Lr19 gene was shown. At the first stage, the development of surface structures was gradually improved, and then, the isolates lost their ability to induce an oxidative burst in stomatal guard cells [51]. It is possible that the elicitor activity of PAMPs was changed at the same time, because after plant treatment with salicylic, succinic acids or benzothiadiazole (an inducer of defense PR-proteins biosynthesis), the ROS generation was increased, and the resistance of the NHR-type was restored [74,75]. The isolate most compatible with cv. Bacanora mainly died after penetration into tissues at different stages—before penetration into cells or after the development of small colonies (with and without HR). Large colonies with pustules were rare, which indicates an extremely unstable interaction between Pgt and plants and partial expression of nonhost mechanisms in the tissues. Previously, similar disrupted interactions of P. triticina with nonhosts T. monococcum and T. timopheevii were shown [76,77]. The need to accumulate a set of genes for the gradual adaptation of rust fungi to the properties of plants with genes introgressed from distant species may explain their durable resistance to diseases.

The theory of nonhost resistance is currently incomplete. However, as the biology of pathogens is studied and the structure and functions of plant genes are clarified, the reasons for incompatibility at certain pathogenesis stages are becoming clear. Now it is known that parasitic fungi perceive physical and chemical properties of plants as stimuli for development [63,78]. It is assumed that the non-compliance of stimuli with needs is the main reason for the suppression of development of non-pathogenic microorganisms and non-specialized forms on the nonhost species [67]. Upon receiving a complex of signals from plants, the pathogen signaling systems (MAPK, cAMP, etc.) are triggered [79]; as a result, the formation and differentiation of infection structures are induced [78,80].

The value of preformed constitutional barriers for the induction of infection structures is shown in some pathosystems. Fungi recognize the surface structural features and the shape of stomata with the help of mechanoreceptors located on the apices of the growing tubes [80]. In the spore adhesion and growing tube attachment to the plant surface, the rust enzymes specialized to waxy cutin (cutinase and esterase) are involved. At the same time, the products of cuticle cleavage serve as signals to identify the host and to induce the gene expression and morphogenesis of infection structures [81]. For the example of powdery mildew fungus B. graminis f. sp. hordei, it was shown that barley wax containing aldehyde C26 stimulated the differentiation of appressoria, and cabbage wax with a high content of aldehydes C28 and C30 suppressed them [82,83]. Differentiation of growing tubes of the alfalfa rust fungus was suppressed by an inhibitor IRG1 (Inhibitor of rust germ tube differentiation 1) associated with the expression of the key wax biosynthesis gene PALM1 (Palmate-like pentafoliata1) [84]. After the penetration of rust fungi into tissues, the development of HMCs and haustoria can be induced by host compounds, including cell wall carbohydrate components and gaseous leaf secretions (nonanal, decanol and hexenyl acetate) [85,86,87,88]. For the example of the Lr23 gene, which is widely distributed in wheat cultivars (from T. turgidum) [89], it was shown that its residual effect was associated with the suppression of the HMCs formation; as a result, a significant portion of the colonies died at different stages of development from starvation without HR [90,91]. A receptor-like kinase LEMK1 (receptor-like kinase) was found in barley, involved in NHR to B. graminis f. p. tritici. Overexpression of the HvLEMK1 gene led to suppression of the formation of fungal haustorium in epidermal cells in transgenic wheat plants [92].

The studies of the NHR genetic control to heterologous (specialized to other species) rust fungi have shown that resistance was inherited polygenically. The barley NHR to heterologous rust fungi was controlled by a set of QTLs with varying effectiveness and overlapping specificity. Most QTLs determine resistance to 1 or 2 heterologous fungi, but some of them defend against more forms [93]. Wheat resistance to heterologous P. striiformis f. sp. hordei was controlled by four QTLs: two determined a strong effect, and two, a weak one [94]. For barley, resistance to Pgt was controlled by five QTLs, of which two were protected only from the wheat form, and others from more heterologous rust fungi [95]. QTLs can control pre- and post-haustorial resistance [96]. Currently, the genes of related cereals are intensively used to protect wheat from rust diseases. For breeding, it is desirable to use QTls and their combinations with strong defense effects. The information we have obtained about the peculiarities of the rye Sr31 gene action can be used to select promising forms with different resistance mechanisms among the interspecies progenies and breeding material.

5. Conclusions

The genetic resources of S. cereale have been little used to protect wheat. Only two genes, Sr31 and Sr50 (Sr31 allele), have been transferred to its genome [97,98]. The results we obtained showed that rye was immune or high resistant to stem rust on infection background of the aggressive Omsk Pgt population. Cytological studies have shown that S. cereale inhibited the development of Pgt on plant surfaces. The rye cv. Petkus (donor of Sr31) most suppressed the appressoria morphogenesis necessary to penetrate into plant tissues. The remaining rye cultivars inhibited Pgt development on the surface at other stages and are of interest as sources of resistance to stem rust.

Among the tested common wheat with the Sr31 gene, cvs. Kavkaz, Seri 82 and Bacanora and line TcLr26/Sr31 were highly resistant to stem rust, and the damage of cv. PWB343 varied from medium to weak. In wheat cultivars, the Sr31 gene suppressed the orientation of germ tubes and the appressoria formation to a lesser extent, but prevented the fungal penetration into tissues by ROS generation in stomatal guard cells. Quantitative differences in the Sr31 gene’s effect on the cultivars were noted. The cvs. Seri 82 and Bacanora and line TcLr26/Sr31 suppressed Pgt development more than cv. PWB343. Probably, the rye’s preinvasion resistance to Pgt was mainly determined by preformed chemical barriers, which were partially transferred to wheat with translocation 1BL/1RS. It is of interest to clarify the molecular basis of the Sr31 gene’s action, and to search for new donors and genes with similar effects.

It is shown that, to overcome the resistance, the fungi have to adapt step-by-step to the properties of plant surface and tissues, which may explain the durable effectiveness of the Sr31 gene. The revealed features of the rye Sr31 gene action may be used as a guide for the selection of promising rust-resistant samples during distant hybridization and breeding wheat cultivars with multi-layered defense.