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.

The OsBSK1-2-MAPK module regulates blast resistance in rice

Receptor-like cytoplasmic kinase OsBSK1-2 was reported to play an important role in regulation of response to rice blast,but the signaling pathway remained unknown.In this study,we identified OsMAPKKK18 and previously uncharacterized MAPKKKs OsMAPKKK16 and OsMAPKKK19 that interact with OsBSK1-2.Expression of all three MAPKKKs was induced by Magnaporthe oryzae infection,and all three induced cell death when transiently expressed in Nicotiana benthamiana leaves.Knockout of OsMAPKKK16 and OsMAPKKK18 compromised blast resistance and overexpression of OsMAPKKK19 increased blast resistance,indicating that all three MAPKKKs are involved in regulation of rice blast response.Furthermore,both OsMAPKKK16 and OsMAPKKK19 interacted with and phosphorylated OsMKK4 and OsMKK5,and chitin-induced MAPK activation was suppressed in osmapkkk16 and osbsk1-2 mutants.OsMAPKKK18 was earlier reported to interact with and phosphorylate OsMKK4 and affect chitin-induced MAPK activation,suggesting that OsBSK1-2 is involved in regulation of immunity through multiple MAPK signaling pathways.Unlike BSK1 in Arabidopsis,OsBSK1-2 was not involved in response to avirulent M.oryzae strains.Taken together,our results revealed important roles of OsMAPKKK16/18/19 and a OsBSK1-2-OsMAPKKK16/18/19-OsMKK4/5 module in regulating response to rice blast.

Effects of β-Amino Butyric Acid Induced Rice Blast Resistance on Reactive Oxygen Metabolism

[Objective] This study was to understand the effects of β-Amino butyric acid(abbreviated as BABA) induced rice blast resistance on reactive oxygen metabolism. [Method] Using the cultivar Chaochan 2 that is highly susceptible to disease as experimental material, the changes of catalase(CAT), and superoxide dismutase(SOD) and MDA activities in rice treated by BABA were investigated. [Result] In rice plants treated by BABA, the activities of CAT and SOD increased, meanwhile the MDA content also rose to some extent, resulting in the disease resistance to rice blast. [Conclusion] By influencing reactive oxygen metabolism, BABA endows rice plants with resistance to rice blast. BABA is safe to environment and has highly resistance-inducing capacity, it could be generalized in production.

Genotypic Analysis of Rice Blast Resistance Genes Pi-ta and Pi-b in Japonica Rice Varieties and Lines in Jiangsu Province

Pi-ta and Pi-b, the first cloned rice blast resistant genes, have been wide- ly used in rice blast resistance breeding for their lasting and stable resistance. To define the distribution of Pi-ta and Pi-b in japonica rice in Jiangsu, the genotypes of resistance genes Pi-ta and Pi-b in 40 varieties and 665 new lines were detected using functional markers of Pi-ta/pi-ta and Pi-b^pi-b alleles. The results showed that the resistance alleles of Pi-ta and Pi-b were widely spread in japonica rice varieties, and the distribution frequency of Pi-b was higher than that of Pi-ta. Most of the Lianjing serial varieties didn＇t carry the two resistance genes, but the two resistance genes were widely distributed in Wujing serial varieties. There was no significant dif- ference in distribution frequency of Pi-ta between new lines and commercial vari- eties. However, the distribution frequency of Pi-b in new lines was higher than that in commercial varieties. It was indicated that artificial selection was conducive to the improvement of distribution frequency of Pi-b in rice varieties. Among the 4 genotypes, the distribution frequency of pi-taJPi-b was highest （60.0%）, followed by Pi-ta/ Pi-b （33.5%） and pi-ta/pi-b （3.9%）. The frequency of Pi-taJpi-b was lowest, account- ing for only 2.6%. In terms of source of resistance genes in the four combinations, the resistant allele Pi-ta might be from parents of Wuxiangjing14, Wujing15 or Nanjing44, and Pi-b might come from parents of Wujing13, Wuxiangjing14, Wujing15 or Nanjing44. The analysis on the genotypic frequencies in offspring of the rice vari- eties showed that the resistance genotype of Pi-ta/Pi-b had the highest frequency in the cross combination of Nanjing44//Wujing13/Kantou194.

Establishment and Application of a Multiplex PCR System for the Detection of Blast Resistance Genes Pi-ta and Pi-b in Rice

Rice blast is one of the important diseases in major rice producing areas of China. The main blast resistance genes Pi-ta and Pi-b showed broad-spectrum and durable resistance to rice blast in many rice growing areas of China, which have been widely utilized in rice breeding and commercial production. In this study, on the basis of detection and verification of the genotypes of 22 rice varieties har- boring known blast resistance genes （Pi-ta and Pi-b） and blast susceptibility genes （pi-ta and pi-b）, two multiple PCR systems for these genes were established by us- ing the functional markers of blast resistance genes Pi-ta and Pi-b as well as blast susceptibility genes pi-ta and pi-b, respectively. Specifically, multiple PCR system I could simultaneously detect blast resistance genes Pi-ta and Pi-b, while system II could detect simultaneously blast susceptibility genes pi-ta and pi-b. In addition, the genotypes of 336 high generation breeding materials were detected with these two multiple PCR systems. The results were highly consistent with those of conventional single mark detection, indicating that these two multiplex PCR systems were stable, reliable and time-saving. The established multiplex PCR systems may serve as a rapid and efficient method to identify and screen rice germplasm resources and can be applied in marker-assisted selection to polymerize multiple genes for blast resis- tance in rice breeding.

RICE TRANSFORMATION WITH A PHYTOALEXIN GENE AND BIOASSAY OF THE TRANSGENIC PLANTS

Immature embryos, mature embryos and embryogenic calli of 6 rice (Oryza sativa L.) materials were transformed with particle bombardment. The plasmids pSSVstl and pVE5+ were used, both containing the phytoalexin gene from grapevine coding for stilbene synthase, but driven by 35S and its own promoter respectively. Through resistance selection for G418 (100 to 150 mg/L) or hygromycin (50 mg/L), 54 independent transgenic plants were isolated and further assessed by PCR, Southern blot and Dot blot analyses. The transgenic plants and their progenies were tested for resistance to blast ( Pyricularia oryzae ) and bacterial blight of rice ( Xanthomonas oryzae ). Preliminary results indicated that the stilbene synthase gene could enhance the resistance of transgenic plants and their progenies to both pathogens.

Study on the Occurrence and Epidemic Regularity and Region Division of Rice Blast in Nanchong City

With the aim to improve the level of monitoring and warning as well as the comprehensive control of rice blast disease, and to feasibly reduce the disease threat in Nanchong City, the methods of GPS and GIS, systematical monitoring and field survey, rice blast resistance identification, physiologic races of rice blast monitoring, and meteorological data analysis were performed to study the occurrence and epidemic region division, precise demarcation and occurrence and epidemic regularity of rice blast in Nanchong City. This study first completed the epidemic region division and precise demarcation; first clarified the initial affection（beginning period） locations, occurrence characteristics, epidemic trends and characteristics; explicated the existence of four epidemic peak periods of rice blast in the field, where the damage areas of first peak period played a decisive role during the blast epidemic years; in late May, the cumulative occurrence areas and annual occurrence areas presented higher positive correlation with the correlation coefficient of 0.817;and established a prediction model of occurrence areas per year based on the disease field rate at the end of boot stages and the diseased plant rate at dough stages. The results of investigation on the impact factors investigation of blast disease in Nanchong in recent years suggested that the internal causes were the decrease or loss of blast resistance of the rice cultivars, as well as the increase of physiological races with strong resistance to rice blast and the emergence of new virulent physiologic varieties; the external causes were suitable temperature, too much rainy, and sunlight shortage. Between 2010 and 2015, the short-term forecast accuracy for rice blast in Nanchong was up to 100%, and medium-and long-term forecast accuracy was also up to 98% and 95%, respectively, which increased by 5-15% than that before 1997, thereby making the control effect of rice blast in Nanchong increased by 15-30%.

Comprehensive Prevention and Control Technologies of Rice Blast in Disease Nanchong City

The study was conducted to reduce blast damage, the use of pesticides residue, environment pollution and control costs, and to make a significant contribution to the improvement of grain production, quality and agriculture ecological environment. Over these years, by the methods of systematical monitoring, regular surveys, field investigation, rice blast resistance identification, experiments and meteorological data analysis, the study on comprehensive prevention and control of rice blast in Nanchong City was conducted. The results showed that the rice varieties more sensitive to blast had a higher incidence of severe blast disease. Replacing,varieties with different source of resistance every three to five years and reasonable variety distribution can effectively reduce the prevalence of rice blast. Appropriate treatment of infected rice straw and pathogen, seed disinfection, seedling disinfection, and pesticide application at transplanting and etc. can delay blast occurrence and reduce the damage caused by blast. By analyzing the blast control efficiency of pesticides applied at different growth stages, we found that best control efficiency against blast was achieved by spraying pesticide twice during the whole growth stage, once 3 d before transplanting or 10 d after transplanting; and once at initial heading stage. Spraying 525 g/hm^2 75% tricyclazole was proven to be the best dosage for blast control. However, 375-450 g/hm^2 75% tricyclazole is enough if the blast incidence is not severe, or the rice varieties are slightly susceptible to blast.The control efficiency against leaf blast between 4% kasugamycin and 20% tricyclazole had no significant difference, but was significantly higher than that of 100 billion spores/g of Bacillus subtilis. The control efficiency against neck blast had no significant difference among 4% kasugamycin, 20% tricyclazole and 100 billion spores/g of B. subtilis. 450 g/hm^2 75% tricyclazole had better control efficiency against neck blast than 2 250 g/hm^2 2% 800 million spores/g Jinggangmycin-wax bud bacteria SC, 1 050 g/hm^2 41% kasugamycin-isoprothiolane WP and 900 g/hm^2 41%kasugamycin-isoprothiolane WP. The frequency of severe blast incidence in Nanchong City has reached 50% since 1997. The rice blast disease has been effectively controlled by comprehensive prevention and control technology, reducing the production loss to less than 2%, and pesticides by 4 523 t in total. In 2014, 327000 t rice grains were approved as pollution-free, green and organic agricultural products, indicating that the blast control measures produced great economic, social and ecological values.

Study on the Prediction of Rice Blast Based on the Unbiased GM (1,1) Model

To create a new prediction model, the unbiased GM （1,1） model is optimized by the five-point slide method in this paper. Then, based on the occurrence areas of dce blast in Enshi District during 1995 -2004, the new model and unbiased GM （1, 1 ） model are applied to predict the occurrence areas of rice blast during 2005 -2010. Predicting outcomes show that the prediction accuracy of five-point unbiased sliding optimized GM （1, 1 ） model is higher than the unbiased GM （1,1） model. Finally, combined with the prediction results, the author provides some suggestion for Enshi District in the prevention and control of rice blast in 2010.

Incidence Law and Disease Symptom of Rice Blast and Its Control Technique

The article systematically summarizes the pathogen, incidence law and disease symptom of rice blast, and raises the integrated control technique for the disease, which will provide the certain theoretical basis for the control of the disease, being benefit for the actual production of rice in field.

Application of Broad-spectrum Blast Resistance Gene Pi40 in Breeding of Japonica Rice in Yunnan Plateau

The resistance of a newly bred line 4163 containing the rice blast resistance gene P/40 and another 11 monogenic lines to rice blast in natural disease nurseries was identified. The results showed that 4163 was resistant to rice blast isolates from Yunnan, and the leaf blast resistance levels were at grades 0, 0 and 4 in Baoshan City, Yiliang City and Yuxi City respectively, while the ear blast resistance was at level 3. Meanwhile, the resistance spectrum of Pi40 to 49 representative rice blast isolates from Yunnan Province was studied by indoor inoculation and was compared with another 25 resistance genes. The results revealed that the resistance frequency of Pi40 to the 49 typical isolates reached 87.8%, showing a broader resistance spectrum, so it could be used as a new source of resistance genes in breeding of japonica rice in Yunnan plateau in the future.

Inhibition Effect of Antagonistic Fungal Strain BOS-013 against the Pathogen of Rice Blast (Pyricularia oryzae)

[ ObjeeUve ] To search for the beneficial microorganisms and their metabolic products with control effect against rice Mast, the paper studied the inhibi- tion effect of the fermentation broth of actinomycetes BOS-013 strain against the pathogen of rice blast （Pyricularia oryzae）. [ Method ] With P. oryzae as the target indicator fungus, the inhibition effect of the fermentation broth of BOS-013 strain and its extracts from different solvents against the pathogen were determined using cylinder plate method and filter paper method. [ Result] The results showed that the inhibition rate of the fermentation broth of BOS-013 strain with fungus amount of 4×10^8 cfu/ml against P. oryzae was 92.0% ±0.5% ; the fermentation broth still had inhibition effect against P. oryaae after inactivation under high temperature and high pressure, and the inhibition rate was 60.0%±0.5%. After absorbed by non-polar CAD-45 type macroperous adsorption resin and eluted by 60% etha- nol, the antifungal activity of the fermentation broth was still high, and the diameter of inhibition zone reached 33.0 mm. [ Conclusion] The fermentation broth of BOS-O13 strain had significant inhibition effect against P. oryzae. The results would provide foundation for further clarification of inhibition mechanism of BOS-013 strain fermentation broth against P. oryzae and its practical application.

Resistance Evaluation of Part of Leading Rice Varieties to Rice Blast in Guangxi Province

In order to determine the resistance of leading rice varieties to rice blast in Guangxi, three natural induction rice blast nurseries were established in Cenxi, Sanjiang and Yongfu. The resistance of the 45 leading rice varieties in Guangxi to rice blast was determined under conditions of spray inoculation and natural induction at seedling stage. The results showed that resistance frequencies of the 45 leading rice varieties ranged from 8.33% to 94.44%. Among the 45 tested varieties, there were 8 varieties of which the resistance frequencies were all higher than 80%, accounting for 17.78%; there were 11 varieties of which the resistance frequencies ranged from 50% to 80%, accounting for 24.44%; the resistance frequencies of the remaining varieties were all lower than 50%, accounting for 57.78% of the total tested varieties. The twoconsecutive-year determination results showed none of the 45 leading rice varieties showed resistance to rice blast. In 2010, there were 26, 12 and 7 varieties that were moderately sensitive, sensitive and greatly sensitive to rice blast respectively, accounting for 57.78%, 26.67% and 15.56% of the total tested varieties. In 2011, there were 16, 23 and 12 varieties that were moderately sensitive, sensitive and greatly sensitive to rice blast respectively, accounting for 35.56%, 51.11% and 13.33%. The overall resistance of the 45 leading rice varieties trended to be decreased year by year. So in rice production, they should be selected properly.

Molecular Cloning of Early-expressed cDNAs from Rice in Response to Infection by Rice Blast Fungus Magnaporthe grisea

Compatible and incompatible reactions in rice plants ( Oryza sativa L. cv. Shenxianggen No.4) were resulted from inoculation with two different virulent races of rice blast fungus ( Magnaporthe grisea (Hebert) Barr), and thus an effective infecting system was established between rice plants and the rice blast pathogen. Two cDNA clones that showed induced and temporal patterns in expression in the very early stage in response to infection of the fungus were obtained from the plants by use of differential display. Of the two cDNA clones, Fastresp_a was induced to express in both compatible and incompatible interactions although it was expressed earlier in the former reaction. The second one, Fastresp_b , was only expressed in incompatible interaction. Southern blot analysis of the rice genomic DNA indicated that both of the two clones were from genome of the plant. No significant homology to the two genes was found from the rice gene database. This suggested that they were novel genes in rice and may play important roles in rice resistant response to infection of rice blast fungus.

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.

Enhancing rice resistance to fungal pathogens by transformation with cell wall degrading enzyme genes from Trichoderma atroviride

Three genes encoding for fungal cell wall degrading enzymes (CWDEs), ech42, nag70 and gluc78 from the biocontrol fungus Trichoderma atroviride were inserted into the binary vector pCAMBIA1305.2 singly and in all possible combinations and transformed to rice plants. More than 1800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. The ech42 gene encoding for an endochitinase increased resistance to sheath blight caused by Rhizoctonia solani, while the exochitinase-encoding gene, nag70, had lesser effect. The expression level of endochitinase but exochitinase was correlated with disease resistance. Nevertheless, exochitinase enhanced the effect of endochitinase on disease resistance when the two genes co-expressed in transgenics. Resistance to Magnaporthe grisea was found in all kinds of regenerated plants including that with single gluc78. A few lines expressing either ech42 or nag70 gene were immune to the disease. Transgenic plants are being tested to further evaluate disease resistance at field level. This is the first report of multiple of expression of genes encoding CWDEs from Trichoderma atroviride that result in resistance to blast and sheath blight in rice.

Changes in Antioxidant Enzyme Activities and Malondialdehyde(MDA) Content of Rice with Blast Resistance Induced by Ag-antibiotic 702

The paper was to explore the effect of rice blast resistance induced by Ag-antibiotic 702 on antioxidant enzyme activities and malondialdehyde（ MDA）content. At three-leaf and one-heart stage of Luliangyou 996,15 μg/mL Ag-antibiotic 702 was sprayed,while Validamycin and distilled water were sprayed as positive control and negative control,respectively. The antioxidant enzyme（ SOD,POD,PPO,CAT and PAL） activities and malondialdehyde（ MDA） content in rice leaves were determined every 24 h within 168 h post spraying. Compared with CK,the MDA content and activities of the five antioxidant enzymes increased in Agantibiotic 702 treatment,Validamycin treatment and M. grisea inoculation treatment. The MDA content and SOD,CAT and POD activities in Ag-antibiotic 702 treatment were significantly higher than those in M. grisea inoculation treatment in early stage,and the APX and LOX activities increased more remarkably and rapidly. The induction effect of Ag-antibiotic 702 could reach or exceed that of Validamycin treatment. Ag-antibiotic 702 enhanced the resistance to rice blast because MDA content and antioxidant enzymes activities were improved in rice leaves. Therefore,the increase of rice blast resistance induced by Ag-antibiotic 702 in rice was positively correlated with the increase of MDA content and antioxidant enzymes activities in rice leaves.

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.

Role of LOX3 Gene in Alleviating Adverse Effects of Drought and Pathogens in Rice

Lipoxygenase 3 (LOX3) is a major component of the LOX isozymes in mature rice seeds. To investigate the role of LOX3 gene under stresses, a plant expression vector containing antisense cDNA of LOX3 was constructed. Rice varieties Wuyunjing 7 and Kasalath were transformed by the Agrobacterium-mediated method and transgenic rice plants were generated. PCR and Southern blot results showed that the antisense LOX3 gene was integrated into the rice genome. Analyses of embryo LOX3 deletion and semi-quantitative RT-PCR confirmed the antisense suppression of LOX3 gene in transgenic plants. The T2 antisense plants of LOX3 were sensitive to drought stress, rice blast and bacterial blight compared with non-transgenic plants. These results suggest that the LOX3 gene might function in response to stresses.

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 48 races of Magnaporthe grisea from Yunnan Province. The transgenic rice lines exhibited resistance to more than 72% of isolates inoculated in this experiment, and 38.1% (24 isolates) of them could infect the receptor cultivar Nan29. The results indicated that the transgenic rice lines possessed wide-spectrum resistance against various rice blast races and the resistant spectrum of rice lines were different although some lines derived from same T0 plant. The transgenic rice lines exhibited also high resistance to leaf and neck blast in the disease field evaluation, but not all of resistant lines against leaf blast were resistant to neck blast.