Pathogenicity Analyses of Rice Blast Fungus (Pyricularia oryzae) from Japonica Rice Area of Northeast China

In order to understand the pathogenicity differentiation of rice blast fungus (Pyricularia oryzae Cavara), a total of 206 isolates of P. oryzae were collected from three Japonica rice regions in Jilin Province, northeast China. Pathogenicity test showed that the reaction pattern of 25 monogenic differential varieties (MDVs) of rice (Oryza sativa L.) demonstrated a wide pathogenic diversity among the isolates. Those MDVs harbor 23 resistance (R) genes with the susceptible variety Lijiangxintuanheigu (LTH) as control. Virulent isolates of MDVs harboring R genes Pish, Pit, Pia, Pii, Pik-s, Pik, Pita (two lines), and Pita-2 (two lines) had high frequencies ranging from 80 to 100%, to MDVs harboring R genes Pib, Pi5(t), Pik-m, Pi1, Pik-h, Pik-p, Pi7(t), Piz, Piz-5, and Piz-t showed intermediate frequencies ranging from 40 to 80%, and to MDVs with R genes Pi3, Pi9(t), Pi12(t), Pi19(t) and Pi20(t) presented low frequencies ranging only from 0 to 40%. The U-i-k-z-ta pattern of race-named criteria categorized the 206 isolates into 175 races. Sub-unit U73 for Pib, i7 for Pi3 and Pi5(t), k177 for Pik-m/Pik-h/Pik-p, z17 for Pi9(t), and ta332 for Pi20(t) were crucial on pathogenic differences in regions. Twenty-seven standard differential blast isolates (SDBIs) were selected to characterize resistance in rice accessions. This study could help to build a durable identification system against blast in the Japonica rice area of northeast China and enhance our understanding of the differentiation and diversity of blast races in the world.

Blast isolates have played a critical role in the identification of resistant germplasm in rice breeding.Each standard differential blast isolate (SDBI) has special avirulent characteristics, so SDBIs have been developed and broadly used in resistance breeding programs [13,[18][19][20].Pathogenically diverse isolates with different avirulence gene proportions have been reported in Cambodia [21], West Africa [22], Bangladesh [19], and Vietnam [20].However, there is no report about SDBIs originating from the DVs in the Japonica rice production area of China.
There are two sets of DVs used successively in northeast China, where Japonica rice is produced [23].The first set of DVs has been used since 1976, including 7 DVs of Tetep, Zhenlong13, Sifeng43, Dongnong363, Guandong51, Hejiang18, and LTH [5].Because their genetic backgrounds on R genes are poorly understood, their identified race information has poorly helped blast-resistance breeding.The second set of DVs contains 25 monogenic differential varieties (MDVs), which harbor 23 resistance genes originating from Japonica LTH.Wang et al. [15] were the first to use this set of MDVs, then followed by others [17,18].Nowadays, the second set of MDVs has been applied to evaluate the pathogenic diversity of isolates, exchange resistant germplasm, understand resistance genetics of germplasm by artificial inoculation, and guide resistance breeding.For example, the Japonica variety Qinglin511 is bred from Jiudao44 as a gene donor, which harbors the R gene Piz, and the local elite variety Jijing88 as a recurrent parent.Field selection in each generation is assisted by blast isolate JLavrPiz-20220817 as an SDBI.As a result, Qinglin511 is resistant to blast with high-yielding and super grain quality [24].
It is well known that planting blast-resistance varieties is the most economical, effectual, and environment-friendly method for controlling rice blast [1,2,25].Blast-resistance breeding is based on a suitable understanding of the pathogenic characteristics of local pathogens.Therefore, our goal in this study was to comprehensively understand the pathogenic characteristics of rice blast isolates in Jilin Province so as to serve blast-resistance breeding.In northeast China, including Heilongjiang, Liaoning, and Jilin provinces, plus nearby Russia, Japan, and North Korea, Japonica rice is planted on five million hectares, where blast is the most severe disease [23,26].Therefore, this study focused on rice blast isolates, aiming to (1) understand virulence diversity and isolate frequency in the Japonica rice area of northeast China; (2) clarify predominant pathogenic types; and (3) select SDBIs for screening accessions to a special pathogen.

P. oryzae Single Spore Isolation and Conservation
During 2019-2021, we collected P. oryzae-infected rice panicles and leaves from three regions in Jilin Province, China: the west drought region (Region I), including Song-Yuan and Bai-Cheng; the central semi-humid region (Region II), including Chang-Chun, Ji-Lin, and Si-Ping; and the east semi-mountain humid region (Region III), including Yan-Ji, Tong-Hua, and Liao-Yuan (Figure 1, Table 1).To obtain a single spore of P. oryzae, the collected samples were incubated on moist filter paper in a Petri dish for 24 h with the protocol described by Wang et al. [15].The incubated samples were shaken off on water-agar medium at 25 • C for 24-36 h until single spores could be noticed with long hyphae under the optical microscope.Each single spore was picked up using small hooks and cultured in a PDA slant medium at 25 • C for 5-7 d.Then, a single colony was transferred to rice straw agar medium covered with filter paper of 9 cm in diameter at 25 • C for 10 d.Finally, these cultures of all single isolates carrying mycelia and conidia were stored at −20 • C in sterile glass vials after necessary drying.

Differential Varieties and Growth Conditions
Our study used 25 rice monogenic lines with a highly susceptible check variety, Menggudao (CK).These monogenic differential varieties (MDVs) harbor 23 single R genes, respectively, including IRBLsh-B(Pish and the backcross parent LTH [27][28][29].Seeds of the monogenic lines and LTH were generously provided by Cailin Lei from the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences (ICS-CAAS).LTH is a Japonica-type local variety from Yunnan Province, China, which is broadly susceptible to rice blast isolates.The Menggudao is used as a super-susceptible control variety or induce variety susceptible to blast disease in disease nurseries in China.Five seeds of each MDV and CK were planted in a plastic tray (68 × 34 × 7 cm) containing local clay soil that had been treated with 0.1% carbendazim fungicide for 3 d.The tray was kept at 20 • C for the night and 28 • C for the day for about four weeks until seedlings were at the 4th-5th leaf stage [15].

Inoculation and Evaluation
Each isolate was inoculated on an oat agar medium with a standard method [30].After 2 weeks, each conidial suspension was standardized to a concentration of 1 × 10 5 spores/mL, then 200 mL standardized spore suspension was sprayed to each tray (1 square meter) with seedlings at the 4th-5th leaf stage using an atomizer.The trays were kept in the dark for 16 h before being transferred to a greenhouse at 25-28 • C with 95% relative humidity.At 6-7 d after inoculation, blast reaction was classified on a scale of 0-5, where 0 means no evidence of infection; 1 means brown specks smaller than 0.5 mm, no sporulation; 2 means brown specks about 0.5-1.0mm in diameter, no sporulation; 3 means roundish to elliptical lesion about 1-3 mm in diameter with a gray center surrounded by brown margins, lesions capable of sporulation; 4 means typical spindle-shaped lesions capable of sporulation, longer than 3 mm with necrotic gray centers and water-soaked or reddish brown margins, little or no coalescence of lesions; and 5 means the same as type 4 but with half of one or two leaf blades killed by the coalescence of lesions as described by Hayashi and Fukuta [31] using Menggudao as susceptible check.The inoculation and evaluation were performed twice sequentially for each rice line and each blast isolate, and the most severe lesion type on the leaf was recorded according to the compatible reaction type.Scales 0-2 were classified as R (Resistant) and 3-5 as S (Susceptible).

Characterization of the Blast Isolates
The R and S reaction patterns of each MDV to the blast isolates were characterized according to the U-i-k-z-ta pattern criteria and pathogenic proportion to MDVs [18].The diversity in the collection was measured with Simpson's diversity index [32].The index values range from 0 to 1, where 0 presents no diversity and 1 maximum diversity [19].

Selection of Standard Blast Isolates
A compatible reaction pattern between MDVs and blast isolates was used to select standard blast isolates (SDBIs).An SDBI had no pathogenicity to one of the MDVs but had very strong pathogenicity to all other MDVs.Based on the gene-for-gene theory, there is a corresponding avirulence gene existing in isolates, which could be used to identify the genetic background of rice accessions resistant to P. oryzae according to the interactioncompatible phenotype.Furthermore, we selected some super-virulent isolates for screening super-resistant germplasm.

Dendrogram Construction and Cluster Analysis
Cluster analysis was performed via Ward's [33] hierarchical method, based on the data of infection scores of the 25 DVs and LTH by blast isolates, in the MEGA11 computer program, aligned using clustalw implemented with the maximum likelihood method.
The 206 isolates from three regions were classified into two groups, and Group I was divided into subgroups Ia and Ib (Supplementary Figure S1).Group I had 49 isolates, while Group II had 157 isolates.In Group I, Subgroup Ia had 47 isolates.(Table 1; Figure 1).It was obvious that Group II was the dominant among the three regions.Moreover, there were no isolates belonging to Subgroup Ib in the west region.
Among the three regions, the east region had the largest isolate number (93), followed by the central region (81) and the west region (32).Similarly, among cluster Group II, the east region had the largest isolate number (68), followed by the central region (63) and the west region (26), and the frequencies were 73.12%, 77.18%, and 81.25%, compared to Group I (Table 1).The details of pathogenic characteristics were presented in the dendrogram (Supplementary Figure S2).

Standard Differential Blast Isolates (SDBIs) Characterization
Based on the race type information, we selected 25 blast isolates as standard differential blast isolates (SDBIs) for screening resistance accessions (Table 3).These SDBIs differentiated the 23 kinds of resistance genes among the 25 DVs.Specifically, the strains of LY1, TH11, SP11, and SY15 corresponded to the broad-spectrum resistance genes of Pi19(t), Pi20(t), Pi12(t), and Pi9(t), respectively (Figure 2d), which could be used to identify the accessions resistant to P. oryzae according to targeted genes.Among them, four isolates, TH20, JL19, LY23, and YJ9, showed different reaction patterns between IRBLta2-Pi and IRBLta2-Re, the same resistance gene Pita-2.And, two isolates, CC13 and SY1, showed different reaction patterns between IRBLta-K1 and IRBLta-CP1, which harbored the same resistance genes Pita (Table 3).Furthermore, two strong virulence isolates, JL27 and SY5, were selected because they were pathogenic to all MDVs harboring all resistance genes (Table 3).The existence of super-virulent isolates indicated a severe threat to rice production.Meanwhile, super-virulent isolates could be used to digest some broad-spectrum resistant accessions to fill special shortcomings in the region.
We divided the identified isolates into two groups (I and II) based on their reaction pattern on 25 MDVs.Those predominant races had high frequencies of reaction types.The frequency variation of blast isolates to DVs is essential for race diversity.For example, in race Subgroup Ib, the U63, the major difference from IRBLb-B(Pib), occurred twice in total, and the frequency was 66.7%.This was the major reason attributable to the Ib group.Similarly, the presence and absence of U53, z07, and k177 were major differences between Group I and Group II.Altogether, the diversity of all the major reaction types corresponded to the distribution of isolates of the cluster.The diversity index of DVs Groups z and k, which was calculated by the Simpson method [32], was higher than that of the other three groups.The diversity of avirulence genes for the blast isolates was important to differentiate the cluster groups.

Figure 1 .
Figure 1.Distribution of blast isolates identified from three regions in Jilin Province, northeast China.

Figure 1 .
Figure 1.Distribution of blast isolates identified from three regions in Jilin Province, northeast China.

Figure 2 .
Figure 2. Pathogenic frequencies of blast isolates to differential varieties (DVs) in Japonica rice samples collected from three regions in Jilin Province, northeast China.(a) frequency of virulent blast isolates in west drought region; (b) frequency of virulent blast isolates in central semi-humid region; (c) frequency of virulent blast isolates in east-semi-mountain humid region; (d) frequency of virulent blast isolates of total isolates.

Figure 2 .
Figure 2. Pathogenic frequencies of blast isolates to differential varieties (DVs) in Japonica rice samples collected from three regions in Jilin Province, northeast China.(a) frequency of virulent blast isolates in west drought region; (b) frequency of virulent blast isolates in central semi-humid region; (c) frequency of virulent blast isolates in east-semi-mountain humid region; (d) frequency of virulent blast isolates of total isolates.

Table 1 .
Blast isolates and frequencies in three regions of Jilin Province, northeast China.

Table 1 .
Blast isolates and frequencies in three regions of Jilin Province, northeast China.

Table 2 .
Reaction types of differential varieties (DVs) in each subgroup.

Table 3 .
Standard differential blast isolates (SDBIs) collected in three regions of Jilin Province.