Rice (Oryza sativa) is one of the major staple food crops for more than half of the human population worldwide [1]. However, a large portion of yield is lost due to agricultural disease and pests annually [2]. Rice blast, caused by the filamentous ascomycete fungusMagnaporthe oryzae (syn. Pyricularia oryzae), occurs in every rice-growing regions in worldwide [3] [4], and poses a major threat to global food security [5] [6]. This pathogen can infect leaves, stems, panicles, nodes, and even roots of rice at the whole stage of development and also infects wheat and other small grains [7] [8] [9]. As genomes of M. oryzae have been sequenced and the data of the genomes is available to the public [10], rice and M. oryzae have been developed as the classical model system for studying the plant-microbe interactions [11].

In many parts of the world, deployment of disease-resistance varieties is an economical and efficient strategy for the management of rice blast [2]. However, M. oryzae often can overcome rice resistance based on race-specific resistant genes [12]. New resistant varieties often lose their resistance within a few years after release to the fields. High genetic variations occur within the field M. oryzae populations, especially in the pathogenic traits i.e., secreted pathogen avirulence or effectors evolve rapidly to counteract plant defense, maybe a major challenge to control rice blast disease [13].

The advent of whole-genome sequencing technology and the subsequent deployment of the next generation sequencing (NGS) technology, genome re-sequencing, comparative studies, and the deployment of PacBio sequencing technology, has been reported in M. oryzae in the last decade [14] [15]. More than 200 isolates of M. oryzae have been successfully generated genome assemblies using high throughput genome sequencing technologies [10] [14] [15]. The genome of the laboratory M. oryzae strain 70 - 15 was the first to be sequenced among plant-pathogenic fungi using the Sanger sequencing method [10]. Subsequently, multiple M. oryzae field isolates have been sequenced using NGS technology. Such as, field isolates from Japan (P131 and Ina168) and China (Y34) were sequenced using 454 sequencing technology [16] [17]. More recently field isolates FJ81278, HN19311, and 98-06 from China, isolates B157, MG01, and RML-29 from India, and isolates R88-002, Ro1-1, and V86010 from the Philippines, and 100 isolates ofM. oryzae (P. oryzae) from China as well as several different regions around the world have been sequenced using Illumina high-throughput sequencing technology [15] [18] [19] [20] [21] [22]. To generate near-complete genome assembly for M. oryzae field isolates and evaluated the possible contribution of TEs to genomic variation events and virulence-associated secreted proteins polymorphism, field isolates FJ81278 and Guy11 have been sequenced by PacBio sequencing technology [14]. The comparison genomics analysis reported some isolate-specific genomic regions, genes under diversifying selection, hundreds of unique genes, and the specific events in the evolution of field populations of M. oryzae [15] [17]. These reports suggest the NGS technology is a powerful tool to elucidate the molecular basis of virulence, study pathogen genome variation, and provide a new method for cloning novel avirulence genes.

Genome-wide studies of more blast field isolates and identification of novel Avr effectors will help researchers to further disclose the mechanism of pathogen and plant co-evolution. QJ08-2006, QJ10-10, and QJ10-3001 were three field M. oryzae isolates in South China, which showed different pathogenicity on a released resistant variety Xin-Yin-Zhan. In this study, we investigated the genomic variations and DNA polymorphisms of three M. oryzae field isolate by NGS technology in an attempt to understand the genomic variation within field isolates after release of the resistant variety. This work will be useful for breeders and pathologists to understand the variation of M. oryzae virulences and develop strategies to improve the resistance of varieties against rice blast.

In several eukaryotic organisms, comparative analysis of multiple genomes of the same species has been used to improve assembly and annotation and to identify genome variations [31] [32] [33]. The rice blast fungus is well-known for its natural genetic variation in the field, resulting in the emergence of new epidemics threatening to world food production [2] [7]. Genomic comparative analyses of multiple genomics in the same pathogens not only could improve the steady of the genome assembly and annotation, and the identification of variations in the plant pathogens’ genome, but also open a door to explore new virulence mechanism of the pathogen for effective plant disease control [20]. In this study, we sequenced three field isolates of M. oryzae from Southern China. These three field isolates were sampled from the varieties Ma-Ba-Yin-Zhan or Xin-Yin-Zhan, which have been cultivated in the rice area of northern Guangdong for a long time [30]. Genome analysis indicated that these re-sequenced field isolates were much more closely related to each other, the overall genome content and composition were similar among these three isolates, indicated that the rice blast fungus maintained a stable genomic mutation under the pressure of resistant varieties selection.

By the pathotype assays, we found QJ08-2006 was avirulent to the rice variety Xin-Yin-Zhan, while both the strain QJ10-10 and QJ10-3001 were impressively virulent to Xin-Yin-Zhan [30]. Comparative genomics found that the sequenced strains have their unique sequences, suggesting some pathogenic-related sequences may exist outside core genome regions [19]. The whole-genome re-sequenced results showed that these three strains showed quite similar features, e.g., genome size, number of predicted genes, and secreted protein genes, showed that they have a closer relationship with each other. And there were as many as 11,076 identical genes in these three isolates and contained only a few unique genes in each isolate (Figure 1). Among them, a small number of the secreted proteins were identified from the unique gene set: 16 (5.78%) in QJ08-2006, 31 (11.74%) in QJ10-10, and 19 (8.92%) in QJ10-3001, showing few of the secreted proteins present in the unique gene set. These data suggested that little genetic differences exist between the field isolates QJ08-2006, QJ10-10, and QJ10-3001.

Yoshida et al. (2009) observed that the presence or absence of effector gene polymorphisms were often associated with unstable genomic regions near the chromosome ends. It was therefore hypothesized that isolate-specific regions located at the chromosomal ends supplied new effectors and pathogenicity-related factors to drive genome evolution. The dynamic adaptation of M. oryzae may be primarily achieved by the deletion or recovery of Avr genes [34]. Such hypotheses indicate that further genome sequencing of multiple M. oryzae isolates is warranted for characterizing rice pathogenic strains [16]. Most of the cloned Avr genes of M. oryzae reflected the important role of transposable elements (TEs) in variations in pathogenicity and the loss of function of Avr genes in M. oryzae [14] [21] [35]. In our study, the effector AvrPib also showed polymorphisms in these three re-sequenced isolates. The isolates QJ10-10, and QJ10-3001 had an insertion of a Pot3 element in the promoter of theAvrPib gene, which has led to a gain in virulence towards rice varieties carrying the R gene, Pib [30]. The Pot3 element inserted in the AvrPib promoter of QJ10-10 and QJ10-3001 suggested this gene is undergoing positive selection, which probably enables these isolates to overcome the resistant rice varieties that harbor AvrPib alleles recently. The loss of function of AvrPib was also identified in 300 isolates collected in five different rice-growing areas in China, and a Pot3 insertion in various positions of −260 bp, −236 bp, −128 bp, −52 bp, −22 bp, and +170 bp, leading to the loss of avirulence function of AvrPib in the five populations [36]. In the 248 virulent isolates collected from a disease hotspot in Philippine, Pot3 was present at the AvrPib region of −304 bp, −125 bp, and +169 bp in each of avrPib-H1, −H2, and −H3 isolates [37]. Similarly, some researchers reported that the diversification in the genome location of Avr-Pita was the consequence of recognition by Pita in rice [12] [34] [38].

To date, only 12Avr genes have been cloned in M. oryzae. By contrast, more than 28 R genes in rice controlling race- and not race-specific resistance to M. oryzae have been cloned [39] [40]. In the traditional “gene-for-gene” model, resistance (R) genes in the host specifically recognize corresponding avirulence (Avr) genes of the pathogens. Recognition is followed by triggering a hypersensitive response (HR) [41]. Thus, there were a lot of potentials to identify and cloneAvr genes in M. oryzae. Before 2009, only the PWL family of three members PWL1, PWL3and PWL4 were cloned by homology to PWL2 [42], other virulence genes mainly were cloned by map-based cloning, such as PWL2, Avr-Pita, ACE1, Avr1-CO39, Avr-Pizt, Avr-Pia, Avr-Pii, AvrPib [12] [35] [36] [43] [44]. With the rapid development of biotechnology and high-throughput sequencing technology, Avr-Piks/Km/Kp, Avr-Pi9and Avr-Pi54 were cloned by using the method of comparative genomics. Due to the instability of the rice blast fungus genome, and that Avr proteins lack conserved domains, the map-based cloning, homology cloning and other traditional cloning strategies were not be effectively utilized. Therefore, whole-genome sequencing of more field blast isolates could accelerate cloning more Avr genes and obtain more information about effector proteins.

Overall, this study provided a systematic genomic analysis of the rice blast field isolates QJ08-2006, QJ10-10, and QJ10-3001, collected from the same field in different years in southern China. Based on the analysis of the three isolates genome, gain, loss and changes of isolate-specific sequences will lead to variances of pathogen virulence. We found that under resistant rice selection pressure for a long time, the genome of avirulent isolate and virulent isolate was very similar, while the changes tend to take place in the genetic variation of avirulence genes corresponding to their resistance genes.

This work was supported by grants from Natural Science Foundation of Guangdong Province (2020A1515011213) and the National key R & D project (2016YFD0300707), Modern Agricultural Industry Technology System (CARS-01-32; 2020KJ105), Guangdong Provincial Science and Technology Project (2019B020217003), and Project of Guangzhou Science Plan (202002030001).

The authors declare no conflicts of interest regarding the publication of this paper.

Chen, K.L., Feng, J.Q., Chen, S., Su, J., Yang, J.Y., Wang, C.Y., Feng, A.Q., Chen, B., Zhu, X.Y. and Wang, W.J. (2021) Comparative Analysis of the Genomes of Three Field Isolates of the Rice Blast Fungus Magnaporthe oryzae from Southern China. Agricultural Sciences, 12, 713-725. https://doi.org/10.4236/as.2021.127046