Global characterization of OsPIP aquaporins reveals that the H_(2)O_(2)transporter OsPIP2;6 increases resistance to rice blast

Plasma membrane intrinsic proteins(PIPs)are conserved plant aquaporins that transport small molecules across the plasma membrane to trigger instant stress responses and maintain cellular homeostasis under biotic and abiotic stress.To elucidate their roles in plant immunity to pathogen attack,we characterized the expression patterns,subcellular localizations,and H_(2)O_(2)-transport ability of 11 OsPIPs in rice(Oryza sativa),and identified OsPIP2;6 as necessary for rice disease resistance.OsPIP2;6 resides on the plasma membrane and facilitates cytoplasmic import of the immune signaling molecule H_(2)O_(2).Knockout of OsPIP2;6 increases rice susceptibility to Magnaporthe oryzae,indicating a positive function in plant immunity.OsPIP2;6 interacts with OsPIP2;2,which has been reported to increase rice resistance to pathogens via H_(2)O_(2)transport.Our findings suggest that OsPIP2;6 cooperates with OsPIP2;2 as a defense signal transporter complex during plant–pathogen interaction.

Application of UAV-Based Imaging and Deep Learning in Assessment of Rice Blast Resistance

Rice blast is regarded as one of the major diseases of rice.Screening rice genotypes with high resistance to rice blast is a key strategy for ensuring global food security.Unmanned aerial vehicles(UAV)-based imaging,coupled with deep learning,can acquire high-throughput imagery related to rice blast infection.In this study,we developed a segmented detection model(called RiceblastSegMask)for rice blast detection and resistance evaluation.The feasibility of different backbones and target detection models was further investigated.RiceblastSegMask is a two-stage instance segmentation model,comprising an image-denoising backbone network,a feature pyramid,a trinomial tree fine-grained feature extraction combination network,and an image pixel codec module.The results showed that the model combining the image-denoising and fine-grained feature extraction based on the Swin Transformer and the feature pixel matching feature labels with the trinomial tree recursive algorithm performed the best.The overall accuracy for instance segmentation of RiceblastSegMask reached 97.56%,and it demonstrated a satisfactory accuracy of 90.29%for grading unique resistance to rice blast.These results indicated that low-altitude remote sensing using UAV,in conjunction with the proposed RiceblastSegMask model,can efficiently calculate the extent of rice blast infection,offering a new phenotypic tool for evaluating rice blast resistance on a field scale in rice breeding programs.

Preventive and Therapeutic Effects of New Fungicides on Rice Blast

[Objectives]The paper was to identify the characteristics and the potency of new fungicides to rice blast disease,and study their preventive and therapeutic effects on the disease.[Methods]Six new and conventional fungicides with different mechanisms of action were selected to compare their preventive and therapeutic effects on rice seedling blast in greenhouse.[Results]The agents which showed to be highly efficacious in blast prevention(greater than 90%)were 25%pyraclostrobin EC,20%fenoxanil SC,75%tricyclazole WP and 50%prochloraz-manganese WP sprayed at the dosages of 112.5-150,180-360,270-450 and 450-600 g/ha,respectively.The therapeutic effect of new fungicides 25%pyraclostrobin EC and 50%prochloraz-manganese WP on rice blast were above 90%when the dosages were 90-150 and 360-600 g/ha,respectively.The therapeutic effect of the conventional fungicide 40%isoprothiolane EC on rice blast was 69.04%when the dosage was 480 g/ha,while those of other fungicides were worse.[Conclusions]Both 25%pyraclostrobin EC and 50%prochloraz-manganese WP have good preventive and therapeutic effects on rice blast,which will provide a reference for the use of these fungicides in the field.

Strategy for Use of Rice Blast Resistance Genes in Rice Molecular Breeding

Rice blast is one of the most destructive diseases affecting rice production worldwide.The development and rational use of resistant varieties has been the most effective and economical measure to control blast.In this review,we summarized the cloning and utilization of rice blast resistance genes,such as Pi1,Pi2,Pi9,Pi54,Pigm and Piz-t.We concluded that three main problems in the current breeding of rice blast resistance are:availability of few R(resistance)genes that confer resistance to both seedling and panicle blast,the resistance effect of pyramided lines is not the result of a simple accumulation of resistance spectrum,and only a few R genes have been successfully used for molecular breeding.Therefore,novel utilization strategies for rice blast R genes in molecular breeding were proposed,such as accurately understanding the utilization of R genes in main modern rice varieties,creating a core resistant germplasm with excellent comprehensive traits,screening and utilizing broadspectrum and durable resistance gene combinations.Lastly,the trends and possible development direction of blast resistance improvement were also discussed,including new genes regulating resistance identified via GWAS(genome-wide association study)and improving rice blast resistance using genetic editing.

Effects of Silicon Fertilizer on Rice Blast (Magnaporthe oryzae) Green Control, Rice Growth and Yield

Three doses of silicon fertilizer and seven dominant rice varieties were selected to study the effects of silicon fertilizer on rice blast(Magnaporthe oryzae)green control,rice growth and yield.The results showed application of silicon fertilizer had certain control effect on panicle blast,and was beneficial for rice growth and yield.However,application effect varied among different varieties,and application of silicon fertilizer at the dose of 225 kg/hm2 received the best effect.Applying silicon fertilizer to control rice blast is safe and environmental-friendly,which is beneficial to the production of green food production.

Improving Rice Blast Resistance by Mining Broad-Spectrum Resistance Genes at Pik Locus

Magnaporthe oryzae is known for its genetic diversity and pathogenic variability,leading to rapid breakdown of resistance in rice.Incorporating multiple broad-spectrum blast resistance genes into rice cultivars would extend disease resistance longevity.Effective resistance breeding in rice therefore requires continual enrichment of the reservoir of resistance genes and alleles.We conducted a large-scale screen of rice blast resistance in about 2000 rice accessions.Among them,247 accessions showed at least medium resistance to the natural infection of rice blast and 7 novel Pik alleles were identified from them.Variations in gene sequences were then correlated with the phenotypic trait to enable the identification of favorable alleles.Among the seven novel Pik alleles,the resistant rate of Pik-R0/ME/7017 donors was greater than 80%,and the disease score was less than 3.Through molecular marker-assisted backcross breeding,we successfully transferred the three Pik alleles,Pik-R0/ME/7017,into an elite cultivated line Kongyu 131 to obtain BC_(3)F_(2)lines,which showed enhanced resistance to rice blast compared with the recurrent parent.Assessment of these near-isogenic lines in the greenhouse using 31 isolates of M.oryzae from Heilongjiang Province of China revealed that the resistant levels of the BC_(3)F_(2)lines with Pik-R0/ME/7017 were significantly higher than those of the established cloned resistance genes Pik-m and Pi1.Exploring such alleles will enrich our gene library for resistance to rice blast.

Genome-Wide Analysis of von Willebrand Factor A Gene Family in Rice for Its Role in Imparting Biotic Stress Resistance with Emphasis on Rice Blast Disease

von Willebrand factor A(vWA)genes are well characterized in humans except for few BONZAI genes,but the vWA genes are least explored in plants.Considering the novelty and vital role of vWA genes,this study aimed at characterization of vWA superfamily in rice.Rice genome was found to have 40 vWA genes distributed across all the 12 chromosomes,and 20 of the 40 vWA genes were unique while the remaining shared large fragment similarities with each other,indicating gene duplication.In addition to vWA domain,vWA proteins possess other different motifs or domains,such as ubiquitin interacting motif in protein degradation pathway,and RING finger in protein-protein interaction.Expression analysis of vWA genes in available expression data suggested that they probably function in biotic and abiotic stress responses including hormonal response and signaling.The frequency of transposon elements in the entire 3K rice germplasm was negligible except for 9 vWA genes,indicating the importance of these genes in rice.Structural and functional diversities showed that the vWA genes in a blast-resistant rice variety Tetep had huge variations compared to blast-susceptible rice varieties HP2216 and Nipponbare.qRT-PCR analysis of vWA genes in Magnaporthe oryzae infected rice tissues indicated OsvWA9,OsvWA36,OsvWA37 and OsvWA18 as the optimal candidate genes for disease resistance.This is the first attempt to characterize vWA gene family in plant species.

<i>In Vitro</i>Efficacy of Botanicals against Rice Blast Pathogen <i>Magnaporthe oryzae oryzae</i>

Rice is the most staple cereal crop of Bangladesh and rice blast caused by Magnaporthe oryzae oryzae (MoO) has become a major factor limiting rice yield in Bangladesh and throughout the world. Eight botanicals extracted both in water and ethanol namely Kalijira (Nigella sativa), Turmeric (Curcuma longa), Ginger (Zingiber officinalis), Garlic (Allium sativum), Onion (Allium cepa), Neem (Azadirachta indica), Allamanda (Allamanda cathartica) and Aloevera (Aloe vera) were tested against MoO in vitro in the Mycology Laboratory, Department of Plant Pathology, Sher-e-Bangla Agricultural University, Dhaka, Bangladesh. All the botanicals significantly reduced radial growth of the tested pathogen. Maximum mycelia growth inhibition of MoO was achieved with water extract of turmeric (1:1 w/v) and ethanol extracts of neem (1:4 w/v) with 86.57% and 92.62% mycelia growth inhibition at 14 DAI, respectively.

Rice Blast Resistance-associated Genes Based on Different RNA-seq Resources

Rice is the main food crop in Asian countries, and rice blast causes an annual loss of rice yield of 30%-35%. Cultivating rice blast resistant varieties and discovering resistance genes are important means to prevent rice blast. By analyzing the changes in rice transcriptome expression levels before and after infection by Magnaporthe oryzae of different physiological races, we found that 5 transcripts were up-regulated together after infection, Os07 t0125500-01, Os07 t0129300-00, Os07 t0126301-01 and Os07 t0124900-01 belongs to the CAP superfamily, and Os10 t0409400-01 encodes a BURP domain containing protein.

Resistance Evaluation of Dominant Varieties against Rice Blast (Magnaporthe oryzae) and Bacterial Blight (Xanthomonas oryzae pv. oryzae) in Guangzhou, China

The resistance of dominant rice varieties in Guangzhou against rice blast(Magnaporthe oryzae)and bacterial blight(Xanthomonas oryzae pv.oryzae)were evaluated by artificial inoculation and field natural blast nursery identification.Among 11 varieties tested,seven varieties showed resistance to rice blast,accounting for 63.6%;two varieties were highly resistant,accounting for 18.2%;three varieties were resistant,accounting for 27.3%;two varieties were moderately resistant,accounting for 18.2%;four varieties were susceptible(moderately susceptible),accounting for 36.4%.Varieties showed different resistance against the prevailing pathotype IV of bacterial blight;one variety was resistant,accounting for 9.1%;three varieties were moderately resistant,accounting for 27.3%;five varieties were moderately susceptible,accounting for 45.5%;one variety was susceptible,accounting for 9.1%;one variety was highly susceptible,accounting for 9.1%;all varieties tested showed susceptible to the virulent pathotype V.Huanghuazhan,Yuejingsimiao 2 and Fengxiuzhan showed resistance against both bacterial blight(pathotype IV)and rice blast,accounting for 27.3%.It is helpful to reduce the production risk by extending such dominant rice varieties with good resistance to rice blast and bacterial blight.

Advances in Integrated Control Techniques of Rice Blast

Rice blast is the most devastating disease in rice,and it seriously threatens the safety of rice production.Improving the ability of integrated prevention and control of rice blast has always been an important part of ensuring food production safety.In the past decade,scientists have made great progress in the prevention and control techniques of rice blast,which paves the way for the green controlling of rice diseases.In this article,the advances in the intelligentization of rice blast field monitoring techniques,the optimization of prediction and forecast modeling system,the research and development of low-toxic and high-efficiency chemical pesticides,biogenic pesticides and inducers and the regulation of multiple ecological factors including variety and cultivation are reviewed,and the new strategies for green controlling of rice blast based on these techniques are summarized.Further,the problems such as high pesticide prices and pesticide residue faced by rice blast prevention and control and the challenge of slow research and development of low-toxic and economical biogenic pesticides are discussed.Finally,the development direction of green controlling of rice blast based on molecular targets,small interfering RNA(siRNA)and CRISPR/Cas9 technologies is predicted,with a view to guaranteeing the safety of rice production.

Comparative Analysis of the Genomes of Three Field Isolates of the Rice Blast Fungus <i>Magnaporthe oryzae</i>from Southern China

Rice blast caused by Magnaporthe oryzae (M. oryzae) is one of the most destructive diseases, which causes significant rice yield losses and affects global food security. To better understand genetic variations among different isolates of M. oryzae in the nature field, we re-sequenced and analyzed the genomes of three field isolates, QJ08-2006, QJ10-10, and QJ10-3001, which showed distinct pathogenicity on Xin-Yin-Zhan, an elite variety in South China. Genome annotation indicated that these three isolates assemblies have similar genome sizes with 38.4 Mb, 38.3 Mb, and 38.4 Mb, respectively. The QJ08-2006 assembly has 2082 contigs with an N50 of 127.4 kb, the QJ10-10 assembly has 2239 contigs with an N50 of 105.13 kb, the QJ10-3001 assembly has 2025 contigs with an N50 of 133.16 kb. A total of 10,432 genes including 1408 putative secreted protein genes were identified from the annotated isolate QJ08-2006 genome, 10,418 genes including 1410 putative secreted protein genes were identified in QJ10-10, and 10,401 genes including 1420 putative secreted protein genes were identified in QJ10-3001. There are as many as 11,076 identical genes in these three isolates and contained only a few unique genes among three isolates, of which 277 unique genes in QJ08-2006 and 264 unique genes in QJ10-10, and 213 unique genes in QJ10-3001. Most of the predicted secreted protein genes had been identified, and the three re-sequenced strains contained 371, 369, and 387 small Indel, respectively. Avr genes were analyzed in several sequenced Magnaporthe strains, the results revealed that Avr-Pi9 and Avr-Piz-t were present in all the sequenced isolates. The isolates QJ08-2006 contained AvrPib, QJ10-10, and QJ10-3001 had an insertion of a Pot3 element in the promoter of the AvrPib gene. Our results showed that, the rapid dominancy of virulence mutant isolates via clonal propagation displayed in the field after the release of the elite variety Xin-Yin-Zhan.

Genetic Transformation of Rice with Pi-d2 Gene Enhances Resistance to Rice Blast Fungus Magnaporthe oryzae

The gene Pi-d2,conferring gene-for-gene resistance to the Chinese blast strain ZB15,was isolated from a rice variety(Digu) by the map-based cloning strategy.Here,we constructed a control plasmid pZH01-pi-d2tp309(pZH01-tp309) and three different expression constructs,pCB-Pi-d25.3kb(pCB5.3kb),pCB-Pi-d26.3kb(pCB6.3kb) and pZH01-Pi-d22.72kb(pZH01-2.72kb) of Pi-d2,driven by Pi-d2 gene's own promoter or CaMV35S promoter.These constructs were separately introduced into japonica rice varieties Lijiangxintuanhegu,Taipei 309,Nipponbare and Zhonghua 9 through Agrobacterium-mediated transformation.A total of 150 transgenic rice plants were obtained from the regenerated calli selected on hygromycin.PCR,RT-PCR and Southern-blotting assay showed that the gene of interest had been integrated into rice genome and stably inherited.Thirty-five transgenic lines independently derived from T1 progeny were inoculated with the rice blast strain ZB15.Transformants exhibited resistance to rice blast at various levels.The lesions on the transgenic plant leaves were less severe than those on the controls and the resistance level of transgenic plants harboring the gene of interest from three vectors had no difference.The own promoter of Pi-d2,about 2.2 kb or 3.2 kb,had the similar promoter function as CaMV35S.Field evaluation for three successive years supported the results of artificial trial,and some lines with high resistance to rice leaf blast and neck blast were obtained.

Development and Identification of Novel Rice Blast Resistant Sources and Their Characterization Using Molecular Markers

To develop and characterize introgression lines for leaf and neck blast resistance, 326 introgression lines were developed using various accessions of six different AA genome wild species in the genetic background of elite Indian varieties like PR114 and Pusa 44 and were screened for blast resistance. Stringent phenotyping coupled with genotyping using gene based markers led to the identification of four resistant introgression lines, which showed promising resistance and do not possess any of the tested genes. Furthermore, multi-location screening confirmed the field resistance of the four introgression lines to both leaf and neck blast. Molecular characterization of these introgression lines using genome-wide simple sequence repeat markers revealed the presence of small percentage of wild Oryza genome introgrssion. So these lines can be used for mapping and identification of novel leaf and neck blast resistance genes. Thus, these four introgression lines can be considered as new genetic resources for blast resistance.

Analysis of Resistant Spectrum to Rice Blast in Transgenic Rice Lines Introduced Lysozyme Gene from T4 Phage

The receptor cultivar Nan29 and thirty-six T5 rice lines derived from ten T0 generation transgen-ic plants harboring lysozyme gene were challenged in the greenhouse by inoculating 63 isolates belonging to 48races of Magnaporthe grisea from Yunnan Province. The transgenic rice lines exhibited resistance to morethan 72% of isolates inoculated in this experiment, and 38.1% (24 isolates) of them could infect the receptorcultivar Nan29. The results indicated that the transgenic rice lines possessed wide-spectrum resistance againstvarious rice blast races and the resistant spectrum of rice lines were different although some lines derived fromsame T0 plant. The transgenic rice lines exhibited also high resistance to leaf and neck blast in the disease fieldevaluation, but not all of resistant lines against leaf blast were resistant to neck blast.

Race Specificity of Major Rice Blast Resistance Genes to Magnaporthe grisea Isolates Collected from indica Rice in Guangdong, China

Race-specific resistance and field resistance of 30 rice blast resistance monogenic lines derived from different resources were evaluated. The spectra of resistance to 163 Magnaporthe grisea isolates collected from indica rice in Guangdong Province, China ranged from 0.6% to 89.6%. Most of the monogenic lines showed a narrow resistance spectrum and high susceptibility in rice blast area, whereas the lines with Pikh and Pi1(t) had the broad resistance spectra of 89.6% and 82.2% respectively, showing a high and stable blast resistance in fields. According to the cluster analysis of specific resistance to 163 blast isolates tested, the 30 monogenic lines were divided into 15 groups, and based on the principal factor analysis, nine kinds of race-specific resistance were identified. Pik, Piz5, Pi9 and Pish can be used as candidate resistance genes for rice breeding since their specific resistance differed from those of the backbone parents in Guangdong, China. Gene pyramiding of Pikh [or Pi1(t)], Pi9 (or Piz5) and Pish (or Pita2) will be effective to obtain broad-spectrum blast resistance in rice breeding program in Guangdong, China. The strategies for studying and application of rice blast resistance genes were discussed.

Functional genomics in the rice blast fungus to unravel the fungal pathogenicity

A rapidly growing number of successful genome sequencing projects in plant pathogenic fungi greatly increase the demands for tools and methodologies to study fungal pathogenicity at genomic scale. Magnaporthe oryzae is an economically important plant pathogenic fungus whose genome is fully sequenced. Recently we have reported the development and application of functional genomics platform technologies in M. oryzae. This model approach would have many practical ramifications in design and implementation of upcoming functional genomics studies of filamentous fungi aimed at understanding fungal pathogenicity.

The Use of Rice Varietal Diversity for Rice Blast Control

Field experiments of mixed- (intercropping) and pure-planting (monoculture) of four rice vari-eties, representing improved hybrid varieties (Shanyou63 and Shanyou22) and high-quality traditional varie-ties (Huangkenuo and Zigu) from Yunnan Province, were conducted based on their differences in genetic back-ground and agro-economical characteristics. The results demonstrated that the mixed-planting of the hybridrice and high-quality traditional rice varieties had a significantly greater effect on controlling rice blast diseasethan the monocuiture of these varieties, particularly the traditional ones. It is evident for the highly suscepti-ble traditional varieties in mixed-planting to achieve disease control, with significant decreases in blast inci-dences and severity indexes. The blast control efficiency reached up to 83 - 98 % under such planting model.This suggests that an appropriate mixed-planting of rice varieties with diverse genetic background and agro-e-conomical characteristics is an effective approach for rice blast control. In addition, resistance of the tradi-tional rice varieties to lodging was considerably increased in the plots with mixed-planting, compared with theplots with monoculture. The average rate of grain-yield increase ranged from 6.5 to 9.7 % in the plots withmixed-planting.

Rice Blast Resistance of Transgenic Rice Plants with Pi-d2 Gene

Resistance to rice blast of transgenic rice lines harboring rice blast resistance gene Pi-d2 transformed from three different expression vectors of pCB6.3kb, pCB5.3kb and pZH01-2.72kb were analyzed. Nine advanced-generation transgenic rice lines with Pi-d2 gene displayed various resistance to 39 rice blast strains, and the highest disease-resistant frequency reached 91.7%. Four early-generation homozygous transgenic lines with Pi-d2 gene exhibited resistance to more than 81.5% of 58 rice blast strains, showing the characteristic of wide-spectrum resistance. The transgenic embryonic calli selected by the crude toxin of rice blast fungus showed that the callus induction rate of immature embryo from transgenic rice plants decreased as the concentration of crude toxin in the culture medium increased. When the concentration of crude toxin reached 40%, the callus induction rate of immature embryo from transgenic lines was 49.3%, and that of the receptor control was 5%. The disease incidence of neck blast of the transgenic rice lines in fields under induction was 0% to 50%, indicating that the rice blast resistance of transgenic rice lines is much higher than that of the receptor control.

A rapid approach for isolating a single fungal spore from rice blast diseased leaves

Single spore isolation is a fundamental approach in plant pathology and mycology to isolate and identify plant fungal pathogens from diseased samples.However,routine single spore isolation procedure is time-consuming and has a high risk of contamination by other microorganisms.In this study,we developed a rapid approach for isolating a single spore of the fungal pathogen,Pyricularia oryzae,from rice blast diseased leaves in the paddy field with low potential of contamination.First,rice blast leaves with single lesions were selected in the paddy field,and a single lesion was cut out and pressed and dragged gently across the surface of water agar.Next,a germinated single spore with a barely visible piece of agar was cut out of water agar with a dissecting needle under a stereomicroscope at approximately 120-fold magnification.Last,the germinated single spore with water agar was transferred onto oatmeal tomato agar for growth and preservation.Based on our experience,a skilled technician or student can successfully isolate single spore from over 150 independent diseased samples with nearly no contaminations in a single working day.This approach is also suitable for isolating single spore from other fungal disease samples that produce abundant aerial conidia.