Investigation of urediospore morphology, histopathology and epidemiological components on wheat plants infected with UV‐B‐induced mutant strains of Puccinia striiformis f. sp. tritici

Abstract Planting resistant cultivars is the most economical and effective measure to control wheat stripe rust caused by Puccinia striiformis f. sp. tritici (Pst), but the cultivars often lose their resistance due to the emergence of new physiological races. The UV‐B‐irradiated urediospores of the Pst physiological race CYR32 in China were inoculated on wheat cultivar Guinong 22 for screening virulence‐mutant strains. CYR32 and mutant strains (CYR32‐5 and CYR32‐61) before and after UV‐B radiation were used to conduct urediospore morphological and histopathological observations and an investigation of epidemiological components. The results showed that UV‐B radiation affected the urediospore morphology of each strain. UV‐B radiation inhibited urediospore invasion and hyphal elongation, which mainly manifested as decreases in germination rate, quantities of hyphal branches, haustorial mother cells and haustoria and hyphal length. After wheat cultivar Mingxian 169 was inoculated with the UV‐B‐irradiated urediospores, the incubation period was prolonged, and the infection efficiency, lesion expansion rate, total sporulation quantity and area under the disease progress curve were reduced. The results demonstrated that CYR32‐5 and CYR32‐61 may have more tolerance to UV‐B radiation than CYR32. The results are significant for understanding mechanisms of Pst virulence variations and implementing sustainable management of wheat stripe rust.

Histopathological methods can be applied to investigate morphological changes of pathogens and plants at the cell and tissue levels during pathogen-plant interactions, and the studies in this aspect can provide a basis for understanding pathogen infection processes and pathogen-host interactions for disease control.
To lay the foundations for exploring the virulence variation mechanism of Pst and to provide a scientific reference for wheat resistance breeding and control of wheat stripe rust, in this study, UV-B radiation was used to treat Pst urediospores to screen virulence-mutant strains of Pst. Then, the original strain and the obtained virulence-mutant strains before and after UV-B radiation were used to systematically investigate the changes in urediospore morphology, histopathology and epidemiological components of the disease.  Cheng et al. (2014), wheat seedlings were incubated and Pst urediospores were multiplied in an artificial climate chamber (11-13°C, 60%-70% relative humidity, 12 hr light at an intensity of 10,000 lux per day).

| Screening virulence-mutant strains
To improve the probability of screening virulence-mutant strains, according to the UV-B radiation intensities acquired in the epidemic areas where virulence variation of Pst frequently occurs in China during the epidemic of wheat stripe rust (Cheng et al., 2014), a UV-B radiation intensity of 250 μw/cm 2 was applied to irradiate the urediospores of CYR32. Using the method described by Cheng et al. (2014) and Zhao, Cheng, Li, and Wang (2018), the radiation time required for an approximately 90% relative lethal rate (i.e., the relative germination rate was approximately 10%) of the CYR32 urediospores under UV-B radiation was selected as the optimal radiation time for screening virulence-mutant strains. Repeated experiments showed that the relative lethal rate of the CYR32 urediospores was approximately 90% when the radiation time was 95 min. Therefore, the radiation time of 95 min was used as the optimal screening time in this study.
After 30 mg of urediospores of CYR32 were treated by UV-B radiation for 95 min with an intensity of 250 μw/cm 2 , a suspension of the irradiated urediospores (0.6 mg/ml) was prepared with 0.2% Tween 80 solution and then artificially sprayed on 12 pots of Mingxian 169 seedlings, for which the first leaves were fully expanded. When the uredinia on the wheat leaves ruptured, the urediospores were collected and then inoculated artificially on the seedlings of Guinong 22 at the growth stage with the first fully expanded leaves. If stripe rust occurred on the seedlings of Guinong 22, the individual uredinia with infection types clearly different from those of the original strain, CYR32, were selected and then propagated throughout four successive generations using a single uredinium isolation method. A strain with stable infection type in the four successive generations was treated as a virulence-mutant strain. The mutation rate was calculated using the following method.
In this study, the virulence mutation rate was calculated based on the number of germinated urediospores on the surface of wheat leaves. The seedlings of Guinong 22 were inoculated with the urediospores collected from Mingxian 169 infected with the UV-B-irradiated urediospores. At 24 hr post inoculation (hpi), leaf samples of Guinong 22 were collected. After the sampled leaves were treated using the living leaf transparency technique, the number of urediospores and the number of germinated urediospores on each leaf were observed directly using a light microscope at 100× (10 × 10) magnification (three replicates per treatment and at least five views for each replicate), and the average spore germination rate was calculated. A urediospore with a germ tube longer than the diameter of the urediospore was considered germinated. The virulence mutation rate of Pst was calculated with the following formula: where MR is the virulence mutation rate, VMS is the number of virulence-mutant strains (i.e., the number of uredinia with stable infection types in the four successive generations on Guinong 22), NL is the number of leaves sampled from the inoculated wheat plants, NS is the average number of spores on an individual wheat leaf and SGR is the average spore germination rate.
The living leaf transparency was performed according to the following method. Each sampled wheat leaf was cut into segments with a length of approximately 2 cm and then put into a 40 ml fixation solution consisting of 95% ethanol and glacial acetic acid (v:v = 3:1).
After 24 hr of fixation, the color of the leaf segments almost completely faded, and the segments were transferred into a solution that was prepared with 25 g of chloral hydrate and 10 ml of distilled water. After 24 hr, the leaf segments were completely transparent and then dyed for 20-30 min with a methyl blue solution consisting of 20 g phenol, 20 ml lactic acid, 40 ml glycerol, 0.05 g methyl blue and 20 ml distilled water.

| Virulence assessment of Pst strains
Virulence determination of the original strain, CYR32, and the obtained virulence-mutant strains was conducted using the 19 Chinese differential hosts described above. The seeds of 19 Chinese differential hosts and Mingxian 169 were sown in five 10 cm-diameter pots with four cultivars per pot and 6-7 seeds per cultivar. When the first leaves were fully expanded, the seedlings of 19 Chinese differential hosts and Mingxian 169 were inoculated with a 0.5 mg/ml urediospore suspension prepared with 10 mg of fresh urediospores of each Pst strain and 20 ml of 0.2% Tween 80 solution. After 15 days, the symptoms of stripe rust appeared on the inoculated wheat leaves, and the infection type of each strain on each cultivar was assessed according to a 0-9 scale (Line & Qayoum, 1992), with 0-3 as the resistant type, 4-6 as the intermediate type and 7-9 as the susceptible type. Three replicate of virulence testing of each strain were performed.

| Electron microscopic observation of the non-UV-B-irradiated and UV-B-irradiated Pst urediospores
For the original strain, CYR32, and each obtained virulence-mutant strain, morphological observations of the non-UV-B-irradiated urediospores and the treated urediospores with UV-B radiation under the dose for which the relative lethal rate of urediospores was 90% (i.e., UV-B lethal dose 90%, LD 90 ) were performed using a scanning electron microscope (SEM). Using the method described by Cheng et al. (2014) and Zhao et al. (2018), the radiation time required for a 90% relative lethal rate of urediospores of each strain under a UV-B radiation intensity of 150 μw/cm 2 was determined. In this study, 3 mg of harvested fresh urediospores of each Pst strain was irradiated with a UV-B radiation dose of LD 90 , and 3 mg of harvested fresh urediospores of the corresponding strain was treated as the control. For each Pst strain, some non-UV-B-irradiated urediospores or UV-B-irradiated urediospores were evenly scattered on a piece of smoothsurface weighing paper. A piece of adhesive copper foil tape with a length of 2 cm was used to stick urediospores. Then, the urediospores on the tape were sputter-coated with gold and finally observed under the microscope.

| Determination of the epidemiological components after inoculation with the non-UV-Birradiated and UV-B-irradiated urediospores of the Pst strains
To investigate the epidemiological components after inoculation with the non-UV-B-irradiated and UV-B-irradiated urediospores of the original strain, CYR32, and the obtained virulence-mutant strains, three treatments were set based on UV-B radiation doses. For two of the three treatments, the urediospores were irradiated with LD 90 and the radiation dose for which the relative lethal rate of urediospores was 50% (i.e., UV-B lethal dose 50%, LD 50 ) under a UV-B radiation intensity of 150 μw/cm 2 , and these two treatments were recorded as the LD 90 treatment and the LD 50 treatment, respectively. For the other treatment, the urediospores were not irradiated with UV-B, and this treatment was used as the control treatment (CK). The radiation time required for LD 90 of each strain under a UV-B radiation intensity of 150 μw/cm 2 was the same as that used for electron microscopic observation of the UV-B-irradiated urediospores of each strain described above. The radiation time required for LD 50 of each strain under a UV-B radiation intensity of 150 μw/cm 2 was determined using the method described by Cheng et al. (2014).
Three replicate were set for each treatment. For each replicate, a 0.1 mg/ml spore suspension prepared with 1 mg of the non-UV-B-irradiated or UV-B-irradiated fresh urediospores of each Pst strain was sprayed on three pots of the seedlings of Mingxian 169, of which the first leaves were fully expanded. The epidemiological components, F I G U R E 1 Infection types of CYR32 (a) and the two mutant strains CYR32-5 (b) and CYR32-61 (c) on Chinese differential hosts. In each row, the wheat cultivars from left to right were Trigo Eureka (T. E.), Fulhard, Lutesens 128, Mentana, Virgilio, Abbondanza, Early Premium, Funo, Danish 1, Jubilejina 2, Fengchan 3, Lovrin 13, Kangyin 655, Suwon 11, Zhong 4, Lovrin 10, Hybrid 46, Triticum spelta album, Guinong 22 and Mingxian 169 including incubation period, infection efficiency, lesion expansion rate, sporulation quantity and area under the disease progress curve (AUDPC), were investigated using the methods described previously (Cheng et al., 2014) with minor modifications. All significant difference analyses of the epidemiological components were performed with Duncan's multiple range tests at the level of 0.05 using the software IBM SPSS Statistics 21.0 (IBM Corp., Armonk, NY).
In this study, the incubation period referred to the days between the day on which the inoculation was conducted and the day on which the first uredinium ruptured. On the eighth day after inoculation, chlorotic spots appeared on the inoculated wheat leaves, and then daily observation of the wheat leaves was performed at the same time of day at which the inoculation was conducted until the day on which the first uredinium ruptured. Infection efficiency was calculated using the formula IE = IL/(Ns × LA), where IE is the infection efficiency, IL is the number of infection loci on each leaf, Ns is the number of urediospores per unit leaf area and LA is the leaf area. Before inoculation, the leaf area of Mingxian 169 was measured using a portable leaf area meter. When the inoculation was conducted, a Vaseline-coated slide was placed beside the wheat leaves for microscopic examination of the number of urediospores per unit area at a magnification of 400× (10 × 40) in five views. When the disease symptoms appeared, the total number of infection loci on all leaves of a pot of wheat seedlings was investigated, and then the average on each leaf was treated as the value of IL. In this study, the lesion expansion rate was defined as the lesion expansion area per day. To determine the lesion expansion rate, a wheat leaf with only one lesion was selected from each replicate and labeled when the disease symptoms appeared on the inoculated wheat seedlings. The length and width of the lesion were measured, and the multiplication of these two values was performed to obtain the lesion area. The lesion was measured every other day until it stopped expanding. The lesion expansion rate was calculated using the formula LER = [(DLA i + 2 − DLA i )/2]/ DLA i × 100%, where LER is the lesion expansion rate, DLA is the disease lesion area and i is the days post inoculation (dpi). The overall lesion expansion rate across the entire lesion expansion period was calculated based on the lesion area measured for the first time and the area measured when the lesion stopped expanding. To assess sporulation quantity, a wheat leaf with only one lesion was chosen from each replicate and labeled when the inoculated wheat leaves of all treatments started to sporulate, and the urediospores produced on this leaf were collected using a test tube (10 cm in length and 1.2 cm in diameter). A urediospore suspension was prepared with the collected urediospores. Using a pipetting gun, 1 μL of suspension was collected for microscopic counting of the urediospores, and in this way, the urediospores in the 5 μL suspension were counted. The average of the urediospore amounts was treated as the number of urediospores per μL.
Then, the sporulation quantity on the leaf was obtained according to the total volume of the urediospore suspension. The urediospore collections on this labeled leaf were performed every other day until no more spores were produced. The total sporulation quantity on the leaf was calculated by summing the sporulation quantities obtained in all urediospore collections across the entire sporulation period. To determine AUDPC, the disease incidence and disease severity of wheat stripe rust were surveyed every five days after the disease symptoms appeared according to the Rules for Monitoring and Forecast of the Wheat Stripe Rust (Puccinia striiformis West.) (National Standard of the People's Republic China, GB/T 15795-2011). The disease severity was classified as 1%, 5%, 10%, 20%, 40%, 60%, 80% or 100%. The value of AUDPC was calculated according to the method described by Cheng et al. (2014).

| Screening and virulence testing results of mutant strains
After screening strains on the seedlings of Guinong 22, two strains with stable infection types throughout the four successive generations were achieved and treated as the virulence-mutant strains.
The calculated virulence mutation rate was 5.37 × 10 −6 . These two mutant strains were named CYR32-5 and CYR32-61. The virulence determination results of mutant strains CYR32-5 and CYR32-61 on TA B L E 1 Infection types of CYR32, CYR32-5 and CYR32-61 on Chinese differential hosts Chinese differential hosts are shown in Figure 1 and

| Investigation results of the epidemiological components after inoculation with the non-UV-Birradiated and UV-B-irradiated urediospores of the Pst strains
Under a UV-B radiation intensity of 150 μw/cm 2 , the radiation times Results of the total sporulation quantities for strains CYR32, CYR32-5 and CYR32-61 are shown in Table 3. For CYR32, the total sporulation quantity under the LD 90 treatment was significantly different (p < 0.05) from that under the control treatment or that under the LD 50 treatment, but there was no significant difference (p > 0.05) between the total sporulation quantities under the control and LD 50 treatments. For strain CYR32-5, the total sporulation quantity under the LD 50 treatment or the LD 90 treatment was significantly different  There were significant differences (p < 0.05) among the AUDPC values on 22-27 or 17-32 dpi under the three treatments for the strain CYR32-61. The results indicated that with increasing UV-B radiation dose, CYR32-5 may be more sensitive to UV-B radiation than CYR32-61.

| D ISCUSS I ON
The aim of this study was to induce mutagenesis of CYR32 by UV-B radiation, obtain virulence-mutant strains and provide a basis for Consistent with the previous SEM observations of the urediospores of Puccinia species (Littefiled, 2000;Traquair & Kokko, 1983), the non-UV-B-irradiated urediospores of Pst strains CYR32, CYR32-5 and CYR32-61 used in this study were covered with spines.
However, the UV-B-irradiated urediospores of the three strains CYR32, CYR32-5 and CYR32-61 irregularly invaginated, the quantity   Beyond the investigations conducted in this study as described above, further studies should be carried out using the obtained virulence-mutant strains of Pst. Studies on virulence-mutant strains obtained using UV radiation at the molecular level were conducted by Huang et al. (2005) and Wang et al. (2009) using a random amplified polymorphic DNA technique, and the results showed that there were significant differences in DNA polymorphisms between mutant strains and wild-type strains. Furthermore, the draft sequences of two Pst isolates have been reported (Cantu et al., 2011;Zheng et al., 2013). In further studies, in-depth mining of the virulence-related genes in virulence-mutant strains of Pst and screening the mutation sites of the corresponding virulence-related genes can be conducted for exploring the mechanisms of Pst virulence variations and breeding disease-resistant wheat cultivars. In this study, two virulencemutant strains of Pst were obtained by UV-B radiation, and some basis was provided for further studies.

| CON CLUS IONS
In this study, two virulence-mutant strains named CYR32-5 and CYR32-61 were obtained from the original strain, CYR32, via UV-B TA B L E 3 The total sporulation quantities for strains CYR32, CYR32-5 and CYR32-61 treated with different doses of UV-B radiation

CO N FLI C T O F I NTE R E S T S
None declared.