The objective of this study is to investigate pathogen species and dominant pathogenic fungi of rice bakanae disease in Heilongjiang Province, and to provide a basis for precise prevention and control of rice bakanae disease.

In 2023, rice bakanae disease samples were collected from 8 cities and 15 counties (districts) in Heilongjiang Province. A total of 172 single-spore strains were isolated and purified from the samples using tissue separation method and dilution plating method. Morphological identification, combined with multilocus phylogenetic analysis using ITS, RPB2, TEF1-α, LSU, and TUB2, and validation via Koch’s postulates, was used to complete the identification of the pathogen causing rice bakanae disease.

172 strains of the pathogenic fungi causing rice bakanae disease were classified into 31 morphotypes based on their morphological characteristics. Multilocus phylogenetic analysis identified 15 genotypes, and the isolated pathogenic fungi included four species complexes: FFSC, FNSC, FOSC, and FIESC. Among them, the dominant pathogenic fungus was Fusarium fujikuroi in the FFSC. A total of 148 strains were obtained, accounting for 86.05% of the total number of isolated strains, and comprising 4 genotypes and 18 morphotypes. Nine strains of Fusarium madaense were also obtained from this species complex, accounting for 5.23% of the total strains, with 1 genotype and 3 morphotypes. In addition, 1 strain of Fusarium subglutinans was identified, representing 0.58% of the total, with 1 genotype and 1 morphotype. In the FIESC, 2 strains of Fusarium wereldwijsianum were obtained, accounting for 1.16% of the total strains, with 2 genotypes and 2 morphotypes; 7 strains of Fusarium ipomoeae were also identified, representing 4.07% of the total, with 5 genotypes and 5 morphotypes. From each of the FNSC and FOSC, 4 strains of Fusarium arbusti and 1 strain of Fusarium cugenangense were obtained, accounting for 2.33% and 0.58% of the total strains, respectively, with 1 genotype and 1 morphotype for each species. All 7 Fusarium species were pathogenic, but with varying degrees of pathogenicity. The dominant pathogenic fungus, F. fujikuroi, caused rice bud rot and etiolation; F. arbusti induced only rice bud rot; F. ipomoeae, F. subglutinans and F. wereldwijsianum inhibited rice growth; F. madaense and F. cugenangense resulted in rice etiolation.

The pathogens causing rice bakanae disease in Heilongjiang Province include 7 species: F. fujikuroi, F. madaense, F. ipomoeae, F. arbusti, F. wereldwijsianum, F. subglutinans, and F. cugenangense. Obvious differences exist in pathogenic types and geographical distribution depending on the pathogen species. Of these, F. fujikuroi and F. arbusti exhibited the strongest pathogenicity; F. fujikuroi was detected in all regions of Heilongjiang Province and served as the dominant pathogenic fungus.

Rice blast seriously threatens rice production in Heilongjiang Province and the breeding and utilization of anti-blast varieties are the most economical, safe and effective measures to control it. This study explored the resistance of rice varieties in Heilongjiang Province; clarified the resistance effect of disease resistance genes, and provided a basis for the selection and utilization of germplasm resources of rice blast in Heilongjiang Province.

In the autumn of 2018, 134 rice strains were collected from the main rice area in Heilongjiang Province, and the resistance of 50 dominant rice varieties was analyzed. For the reported 35 rice blast resistance genes, a comparison was made between the varieties and positive control varieties. Sequencing results of some positive control varieties were compared with the reference sequence published in the National Centre for Biotechnology Information to analyze the existence of rice blast resistance genes in the corresponding varieties. Through the correlation analysis of gene aggregation type and breed resistance performance, the genotypes related to the resistance performance of rice varieties in Heilongjiang Province were clarified.

Among 50 rice varieties in Heilongjiang Province, Longgeng 20 had the best resistance (R). Longgeng 67, Longken 202, Longgeng 40, Longgeng 31, Longgeng 57 and Longgeng 43 had moderate disease resistance; whereas, 43 varieties such as Jiahe 1 had relatively low resistance. Through the analysis of variety combination resistance, we found that 33 pairs of Longgeng 20 + Longgeng 67 had a high RAC (resistance association coefficient) value, low VAC (virulence association coefficient) value and good combined disease resistance. The matching structure showed potential for application. The identification of resistance genes carried by specific primers showed that Pish, Pi36, Pi33 and Pi-CO39 were detected, and Pi63, Ptr, Pi37, Pi64, pi21, Pi9, Pi54, Pikh, Pia, Pikp, Pi35, Pikm and Pik were between 50%-100% of occurences frequency, indicating that such genes were widely used in rice breeding in Heilongjiang Province. The detection rate of Pita, Pib, Pii, Pi5, Piz-t, Pi50 and Pi2 was between 10% and 50%; Pid2 was detected in in two varieties, and Pigm was detected in Jigeng 88. However, Pit, Pid3, Bsr-d1, Pi25, Pid3-A4, Pi56, Pi1, Pike and Pb1 were not detected in the cultivars, indicating that such genes are less distributed in rice varieties in Heilongjiang Province. The variety genotype analysis found that the varieties carried 12-19 resistant genes, with a total of 58 genotypes, indicating that the test varieties had rich blast resistance gene combination types. By analysis, the single gene and gene polymerization with disease resistance showed that the distribution frequency of Pi2, Piz-t, Pi50, Pi5 and Pii corresponded to frequency of resistance. We found that the more varieties carrying resistance genes, the higher the frequency resistance, and six varieties that carrying Pi2+Piz-t+Pi50+α polymerization types showed resistance.

Our results have demonstrated the following: the resistance of rice germplasm resources in Heilongjiang province is low; combination planting of different varieties could be usefully applied; the distribution of blast resistance genes is different in the participating varieties; Pi2, Piz-t, Pi50, Pi5 and Pii play a leading role in disease resistance, and Pi2+Piz-t+Pi50+α gene polymerization type could contribute to improving rice blast resistance.

The objective of this study is to understand the distribution and variation characteristics of PWL gene family in different Magnaporthe oryzae strains, and to provide a basis for studying the genetic diversity and specificity of different M. oryzae strains.

A total of 8 pairs of specific primers were designed for the promoter region and CDS region of the PWL gene family by referring to the avirulence gene sequence published in NCBI. DNA was extracted from 397 single spore isolates of M. oryzae collected from different regions of Heilongjiang Province and Hainan Province in 2020. PCR amplification of avirulence genes was conducted and the genes were detected by agarose gel electrophoresis. Representative strains from different regions were selected from the test results to sequence and analyze the amplified fragments. The sequencing results were compared with the base and amino acid sequences of the corresponding avirulence gene promoter region and CDS region in NCBI.

In the results of PCR electrophoresis, PWL1 was not detected in all strains. The specific fragments of PWL2, PWL3 and PWL4 were amplified in Heilongjiang Province and Hainan Province, indicating that these three genes were distributed in both provinces and had different distribution frequencies and variation types. Among them, PWL2 had the highest distribution frequency in Heilongjiang Province and Hainan Province, which were 98.14% and 100%, respectively. The distribution frequencies of PWL3 and PWL4 in the two provinces were significantly different. The frequencies of these two genes in Heilongjiang strains were 89.30% and 82.79%, respectively, while in Hainan strains were 5.49%. Through the analysis of avirulence gene combinations, the results showed that the combination types could be divided into six types, namely PWL(f), PWL2, PWL3, PWL2+PWL3, PWL2+PWL4, PWL2+PWL3+PWL4. Among them, Heilongjiang strains contained all combination types, and Hainan strains contained only two, indicating that Heilongjiang strains were more abundant in avirulent genotypes than Hainan strains. By sequencing the PCR products of the PWL gene family, it was found that the PWL gene family had abundant variation sites in the promoter region and CDS region, and was divided into nine types with point mutations and deletions as the main variation types, and the variation types of strains from different populations were specific and consistent. Among them, five variant types PWL2- (1, 2, 3, 4, 5) were detected in PWL2, base sequence changes led to amino acid sequence missense mutations; two mutation types were detected in PWL3 and PWL4, which were PWL3- (1, 2) and PWL4- (1, 2), respectively. Both of them had frameshift mutations that caused changes in the following amino acids.

The distribution and variation types of PWL gene family in M. oryzae strains from different populations have regional differences, and the variation sites are abundant.

By detecting the distribution and variation characteristics of the AVR-Pita family in different Magnaporthe oryzae strains from Heilongjiang Province and Hainan Province, the pathogenic phenotypes of the variation types were understood, which provided a reference for the screening and breeding of disease-resistant germplasm in the region.

Six pairs of specific primers were designed for the promoter region and CDS region by referring to the AVR-Pita family sequence published in NCBI. DNA was extracted from 397 single spore strains of M. oryzae collected from different regions of Heilongjiang Province and Hainan Province in 2020, and PCR amplification was performed. By electrophoresis test results, representative strains from different regions were selected to sequence the amplified fragments. The sequencing results were compared with the corresponding base and amino acid sequences in NCBI, and the avirulence function of different variation types of M. oryzae strains was verified by using rice resistant single gene lines.

In the PCR electrophoresis results, the M. oryzae strains in Heilongjiang Province carried all the genes to be tested, which were widely distributed and had a high detection frequency. However, only AVR-Pita1 and AVR-Pita2 were carried by M. oryzae strains in Hainan Province, and concentrated in low frequency. The results of avirulence gene composition analysis showed that the strains of M. oryzae in Heilongjiang were complex and diverse, and the genotypes were more abundant than those in Hainan. The sequencing results of PCR products showed that the AVR-Pita family was divided into 19 types with point mutation, insertion and deletion as the main variation types, and the variation types of strains from different M. oryzae populations were specific. Ten variation types were detected in AVR-Pita1, among which AVR-Pita1- (1-5) was a unique variation type of M. oryzae strains in Heilongjiang Province, and AVR-Pita1- (6-10) was a unique variation type of M. oryzae strains in Hainan Province. After functional verification of these ten variation types, it was found that the avirulence functions were lost. Eight variation types were detected in AVR-Pita2, among which AVR-Pita2- (1-4) was a unique variation type of M. oryzae strains in Heilongjiang Province, and AVR-Pita2- (5-8) was a unique variation type of M. oryzae strains in Hainan Province. After functional verification, it was found that the avirulence functions were lost. Only one variation type (AVR-Pita3-1) was detected in AVR-Pita3 in Heilongjiang Province.

The AVR-Pita family in Heilongjiang and Hainan populations were mutated alleles. After pathogenicity identification, all mutation types could not be identified by the corresponding resistance genes Pi-ta and Pi-ta². Therefore, the resistance genes Pi-ta and Pi-ta² can be used to polymerize other resistance genes to ensure the disease resistance of varieties in the process of disease resistance breeding and utilization of rice blast in Heilongjiang Province and Hainan Province. At the same time, the distribution and variation types of AVR-Pita family in M. oryzae strains from different geographical sources are specific.

The objective of this study is to identify the pathogenic species and dominant pathogens of rice spikelet rot disease (RSRD) in Heilongjiang, Sichuan and Hainan provinces of China, and to provide reference for accurate prevention and control of RSRD.

RSRD samples were collected from 13 major rice production areas in Heilongjiang, Sichuan and Hainan provinces. A total of 568 strains were isolated and purified by tissue separation and single spore separation methods. Morphological identification methods were performed for each strain, combined with rDNA-ITS sequence analysis and Koch’s rule verification and identification of RSRD pathogens in 13 major rice production areas. The dominant strains and pathogenic characteristics were analyzed.

There were three categories of pathogens of RSRD in Heilongjiang Province, they were Curvularia, Alternaria and Fusarium, and which accounted for 1.10%, 83.43% and 15.47% of the total pathogens, respectively. Alternaria was the dominant strains in Heilongjiang Province. In Hainan Province, there were Curvularia, Fusarium, Nigrospora, Lasiodiplodia and Exserohilum species of RSRD pathogens, which accounted for 1.62%, 89.47%, 0.81%, 1.62% and 6.48% of the total pathogens, respectively, and Fusarium was the dominant strains. There were Curvularia, Alternaria, Fusarium, Nigrospora and Exserohilum species of RSRD pathogens in Sichuan Province, which accounted for 23.57%, 13.47%, 22.86%, 16.43% and 23.57% of the total pathogens, respectively, and no dominant strain. According to the differences of conidia and colony morphology, 23 representative strains were selected, and then rDNA-ITS sequence analysis and Koch’s rule verification were combined. The pathogens of RSRD were identified as C. lunata of the genus Curvularia; A. tenuissima, A. alternata, A. brassicae of the genus Alternaria; F. verticillioides, F. andiyazi, F. equiseti, F. incarnatu, F. chlamydosporum of the genus Fusarium; N. oryzae, N. sphaerica of the genus Nigrospora; L. theobromae of the genus Lasiodiplodia and E. rostratum of the genus Exserohilum. The pathogens of RSRD were inoculated both at booting stage and heading and flowering stage, respectively. It was found that whether japonica or indica rice, the average disease index of pathogen of heading and flowering stage inoculation was higher than that of inoculation at booting stage in Hainan Province; while the pathogens’ average disease index of heading and flowering stage inoculation in Heilongjiang and Sichuan provinces was slightly lower than that at booting stage inoculation.

The pathogens of spikelet rot disease in Heilongjiang, Sichuan and Hainan provinces of China were diverse. The dominant strain was Alternaria in Heilongjiang Province and Fusarium in Hainan Province. The dominant strain was not obvious in Sichuan Province.