The DNA damage repair complex MoMMS21-MoSMC5 is required for infection-related development and pathogenicity of Magnaporthe oryzae

The conserved DNA damage repair complex,MMS21-SMC5/6(Methyl methane sulfonate 21-Structural maintenance of chromosomes 5/6),has been extensively studied in yeast,animals,and plants.However,its role in phytopathogenic fungi,particularly in the highly destructive rice blast fungus Magnaporthe oryzae,remains unknown.In this study,we functionally characterized the homologues of this complex,MoMMS21 and MoSMC5,in M.oryzae.We first demonstrated the importance of DNA damage repair in M.oryzae by showing that the DNA damage inducer phleomycin inhibited vegetative growth,infection-related development and pathogenicity in this fungus.Additionally,we discovered that MoMMS21 and MoSMC5 interacted in the nuclei,suggesting that they also function as a complex in M.oryzae.Gene deletion experiments revealed that both MoMMS21 and MoSMC5 are required for infection-related development and pathogenicity in M.oryzae,while only MoMMS21 deletion affected growth and sensitivity to phleomycin,indicating its specific involvement in DNA damage repair.Overall,our results provide insights into the roles of MoMMS21 and MoSMC5 in M.oryzae,highlighting their functions beyond DNA damage repair.

The Magnaporthe oryzae effector Avr-PikD suppresses rice immunity by inhibiting an LSD1-like transcriptional activator

Avirulence effectors(Avrs),encoded by plant pathogens,can be recognized by plants harboring the corresponding resistance proteins,thereby initiating effector-triggered immunity(ETI).In susceptible plants,however,Avrs can function as effectors,facilitating infection via effector-triggered susceptibility(ETS).Mechanisms of Avr-mediated ETS remain largely unexplored.Here we report that the Magnaporthe oryzae effector Avr-PikD enters rice cells via the canonical cytoplasmic secretion pathway and suppresses rice basal defense.Avr-PikD interacts with an LSD1-like transcriptional activator AKIP30 of rice,and AKIP30 is also a positive regulator of rice immunity,whereas Avr-PikD impedes its nuclear localization and suppresses its transcriptional activity.In summary,M.oryzae delivers Avr-PikD into rice cells to facilitate ETS by inhibiting AKIP30-mediated transcriptional regulation of immune response against M.oryzae.

Molecular Screening of Rice Cultivated in Benin for the Identification of Xanthomonas oryzae Pv. oryzae and Bacterial Leaf Blight Resistance Genes

One of the most devastating diseases of rice worldwide is bacterial blight (BLB) caused by Xanthomonas oryzae pv. Oryzae (Xoo). In Benin, Xoo was first described in 2013 on wild rice Oryzae longistaminata. So far, no study has been done on Beninese Xoo strains. We do not know whether the pathogen has already passed into the rice varieties grown, or if they are exposed to other bacteria. Whereas the use of resistant varieties, carrying resistance genes, is the only highly effective and environmentally friendly way to control this disease, no information is available on these Xoo resistance genes in rice varieties grown in Benin apart from the one we recently. This study aims to identify Beninese Xoo strains, causing BLB and screen rice varieties grown in Benin for the main resistance genes. Diseased rice leaves showing typical symptoms of fire blight collected from different rice fields in the three phytogeographic areas of Benin were analyzed by PCR for Xoo-specific sequence identification. Furthermore, seventy-five collected rice accessions were screened to identify xa5, Xa7, xa13, and Xa21 resistance genes to Xoo. The results reveal that Xanthomonas oryzae was identified in two fields in Banikouara and one in Malanville. On the other hand, Sphingomonas sp. has been identified in several other rice fields in Benin. Forty-seven of seventy-five rice accessions examined (62.66%) carried Xoo resistance genes with 3 (4%) and 40 (53.33%) of xa5 and Xa21 respectively. None of the accessions had either Xa7 or xa13 resistance genes. Three accessions possess both xa5 and Xa21 genes. Isogenic lines IRBB60 and IRBB21, supposed to be a positive control, presented a Xoo sensitivity allele. These results indicate that Xoo has moved from the wild rice variety to the cultivated variety in northern Benin and varietal improvement programs must be implemented with varieties having several resistance genes for the efficient response against a possible BLB pandemic in Benin.

Ecofriendly Management of Wheat Panicle Blast Caused by Magnaporthe oryzae triticum

In this study, three wheat varieties were tested to determine seed germination and the incidence of Magnaporthe oryzae triticum (MoT). Among these varieties, BARI Gom 24 (Prodip) wheat seed exhibited the highest seed germination rate (93%) but also had the highest incidence (30%) of MoT. To manage blast disease in an ecofriendly manner, seven treatments were employed: T1 = Control, T2 = Garlic clove extracts, T3 = Aloe vera leaf extracts, T4 = Black cumin seed extracts, T5 = Neem leaf extracts, T6 = Nativo 75 WG, and T7 = Provax 200 WP. The experiment was conducted using a Randomized Complete Block Design (RCBD) layout with three replications using Prodip wheat variety that exhibited highest MoT infection severity based on laboratory analysis among collected varieties. Data were collected on blast disease incidence (%), disease severity, and various growth and yield parameters of wheat. The experiment’s results indicated that among all the treatments, T7 (Seed treatment with Provax 200 WP) and T5 (Foliar spraying with Neem leaf extract) performed better in controlling blast disease in wheat. The lowest blast disease incidence (%) was observed with T7 (Provax 200 WP), with values of 7.86, 9.86, and 10.19 recorded during the milking stage, soft dough stage, and hard dough stage of wheat, respectively. T5 (Neem leaf extract) also demonstrated a statistically equivalent reduction in blast disease incidence (%). In terms of disease severity, T7 (Seed treatment with Provax 200 WP) showed the lowest values of 1.03, 1.23, and 1.63 during the milking stage, soft dough stage, and hard dough stage of wheat, respectively. Foliar spraying with neem leaf extract also exhibited similar result as of Provax 200 WP regarding panicle blast severity. As a result of these findings, it can be concluded that T5 (Neem leaf extract) is recommended as an ecofriendly management approach for blast disease in wheat.

Synthetical Evaluation on the Qualitative and Quantitative Resistance of Rice Germplasms to Magnaporthe oryzae

[Objectives]This study was conducted to screen out rice resources resistant to rice blast(Magnaporthe oryzae).[Methods]The qualitative and quantitative resistance of 1659 rice resources from 45 countries and regions to rice blast were evaluated by disease nursery in upland condition and the test of the spectrum to rice blast isolates.[Results]There were 292 entries which accounted for 17.6%showed high blast resistance(0 and 1 disease scale),68 entries(counted for 4.1%)showed resistance to blast(3 disease scale);and the number of the entries showed intermediate resistance,intermediate susceptible and susceptible were 208(with the corresponding percentage of 12.5%),471(28.4%),620(37.4%)respectively.Among the tested entries,27 entries including BG1222,BL122,BTX,IR37704-131-2-3-2,and LEBONNET had showed broad-spectrum blast resistance with the resistance frequency of higher than 90%,Quantitative resistance evaluation was conducted on some key resources,and 14 entries,of which are BR27,DRAGO,IR100,QINLIUAI、SERIBU GANTANG,YUEXIANGZHAN and so on,showed good quantitative resistances,and 8 entries had higher quantitative resistances than IR36.[Conclusions]This study provides important blast resistance resources for the local rice breeding program and has a significant value for the discovery of new blast resistance genes and its application in the blast resistance breeding.

Evaluation of in vitro digestibility of Aspergillus oryzae fungal biomass grown on organic residue derived‑VFAs as a promising ruminant feed supplement

Background As demand for high quality animal feed continues to raise,it becomes increasingly important to mini-mize the environmental impact of feed production.An appealing sustainable approach to provide feed fractions is to use organic residues from agro-food industry.In this regard,volatile fatty acids(VFAs)such as acetic,propionic and butyric acids,derived from bioconversion of organic residues can be used as precursors for production of micro-bial protein with ruminant feed inclusion potential.This study aims to investigate the in vitro digestibility of the Asper-gillus oryzae edible fungal biomass cultivated on VFAs-derived from anaerobic digestion of residues.The produced fungal protein biomass,along with hay clover silage and rapeseed meal were subjected to various in vitro assays using two-stage Tilley and Terry(TT),gas,and bag methods to evaluate and compare its digestibility for application in ruminant feed.Results The produced fungal biomass contained a higher crude protein(CP)(41%–49%)and rather similar neutral detergent fiber(NDF)(41%–56%)compared to rapeseed meal.The rumen in vitro dry matter digestibility(IVDMD)of the fungal biomass in the TT method ranged from 82%to 88%(statistically similar to that of the gas method(72%to 85%)).The IVDMD of fungal biomass were up to 26%and 40%greater than that of hay clover silage and rapeseed meal,respectively.The type of substrate and bag method had pronounced effect on the fermentation products(ammonium-N(NH4+-N),total gas and VFAs).Fungal biomass digestion resulted in the highest release of NH4+-N(340–540 mg/L)and the ratio of acetate to propionate ratio(3.5)among subjected substrates.Conclusion The results indicate that gas method can be used as a reliable predictor for IVDMD as well as fermenta-tion products.Furthermore,the high IVDMD and fermentation product observed for Aspergillus oryzae fungal biomass digestion,suggest that the supplementation of fungal biomass will contribute to improving the rumen digestion by providing necessary nitrogen and energy to the ruminant and microbiota.

The auxin transporter OsAUX1 regulates tillering in rice(Oryza sativa)

Tillering is an important agronomic trait of rice(Oryza sativa)that affects the number of effective panicles,thereby affecting yields.The phytohormone auxin plays a key role in tillering.Here we identified the high tillering and semi-dwarf 1(htsd1)mutant with auxin-deficiency root characteristics,such as shortened lateral roots,reduced lateral root density,and enlarged root angles.htsd1 showed reduced sensitivity to auxin,but the external application of indole-3-acetic acid(IAA)inhibited its tillering.We identified the mutated gene in htsd1 as AUXIN1(OsAUX1,LOC_Os01g63770),which encodes an auxin influx transporter.The promoter sequence of OsAUX1 contains many SQUAMOSA PROMOTER BINDING PROTEIN-LIKE(SPL)binding sites,and we demonstrated that SPL7 binds to the OsAUX1 promoter.TEOSINTE BRANCHED1(OsTB1),a key gene that negatively regulates tillering,was significantly downregulated in htsd1.Tillering was enhanced in the OsTB1 knockout mutant,and the external application of IAA inhibited tiller elongation in this mutant.Overexpressing OsTB1 restored the multi-tiller phenotype of htsd1.These results suggest that SPL7 directly binds to the OsAUX1 promoter and regulates tillering in rice by altering OsTB1 expression to modulate auxin signaling.

Irrigation regimes modulate non-structural carbohydrate remobilization and improve grain filling in rice(Oryza sativa L.)by regulating starch metabolism

Recently developed ‘super’ rice cultivars with greater yield potentials often suffer from the problem of poor grain filling, especially in inferior spikelets. Here, we studied the activities of enzymes related to starch metabolism in rice stems and grains, and the microstructures related to carbohydrate accumulation and transportation to investigate the effects of different water regimes on grain filling. Two ‘super’ rice cultivars were grown under two irrigation regimes of well-watered(WW) and alternate wetting and moderate soil drying(AWMD). Compared with the WW treatment,the activities of ADP glucose pyrophosphorylase(AGPase), starch synthase(StSase) and starch branching enzyme(SBE), and the accumulation of non-structural carbohydrates(NSCs) in the stems before heading were significantly improved, and more starch granules were stored in the stems in the AWMD treatment. After heading, the activities of α-amylase, β-amylase, sucrose phosphate synthase(SPS) and sucrose synthase in the synthetic direction(SSs)were increased in the stems to promote the remobilization of NSCs for grain filling under AWMD. During grain filling, the enzymatic activities of sucrose synthase in the cleavage direction(SSc), AGPase, StSase and SBE in the inferior spikelets were increased, which promoted grain filling, especially for the inferior spikelets under AWMD.However, there were no significant differences in vascular microstructures. The grain yield and grain weight could be improved by 13.1 and 7.5%, respectively, by optimizing of the irrigation regime. We concluded that the low activities of key enzymes in carbon metabolism is the key limitation for the poor grain filling, as opposed to the vascular microstructures, and AWMD can increase the amount of NSC accumulation in the stems before heading, improve the utilization rate of NSCs after heading, and increase the grain filling, especially in the inferior spikelets, by altering the activities of key enzymes in carbon metabolism.

培养过程参数对霉菌Rhizopus oryzae IFO细胞催化植物油脂合成生物柴油的影响研究

采用脂肪酶催化可再生动植物油脂合成生物柴油已经成为目前研究的热点,其中利用全细胞催化剂是一个重要的研究方向。文中直接利用霉菌R.oryzaeIFO细胞催化植物油脂甲醇解反应合成生物柴油,系统研究了培养过程参数对细胞生长和该细胞催化剂催化甲醇解反应活性的影响。研究表明,细胞培养过程中所添加的油脂不同,细胞在后续反应中催化特定油脂进行生物柴油制备时所表现出的催化活性也会有所差别;由某种油脂作为碳源得到的细胞催化剂催化对应油脂与甲醇转酯化反应制备生物柴油时,表现出比催化其他油脂和甲醇反应制备生物柴油更高的催化活性。在优化的操作参数(大豆精制油20g/L,蛋白胨70g/L,NaNO31.2g/L,KH2PO41.2g/L,MgSO4·7H2O0.5g/L,培养温度35℃,摇床转速130r/min)下,培养得到的细胞催化剂能有效催化大豆油与甲醇三步转酯化反应,生物柴油(脂肪酸甲酯)最终得率可达到86%。

固定化R.oryzae细胞发酵产胞内脂肪酶及其催化制备生物柴油

采用含胞内脂肪酶的微生物细胞催化油脂原料生产生物柴油是目前生物柴油生产领域的一个新的研究方向。探讨了以聚氨酯泡沫颗粒作为载体固定化R.oryzae细胞对细胞生长与产胞内脂肪酶的影响,并对固定化R.oryzae发酵条件进行了优化,特别对氮源的选用进行了研究。结果表明,选用廉价的豆粉作为有机氮源培养R.oryzae细胞效果较好,与以蛋白胨作为培养基氮源相比,单位培养基所得菌体的总酶活提高至1.55倍,同时,氮源利用率大大提高。进一步考察了有机氮源与无机氮源复合培养R.oryzae细胞的情况,发现豆粉与(NH4)2HPO4复合效果较好。利用正交设计对培养基中各种无机盐成分进行了优化。优化结果为全脂豆粉2%,大豆油3%,MgSO40.035%,K2HPO40.12%,(NH4)2HPO40.10%,上述培养条件下,单位培养基培养菌体胞内脂肪酶活可达6054.2U·L-1培养基。以该条件下培养菌体催化大豆油生产生物柴油,在叔丁醇体系下,反应24h生物柴油得率可达68.5%。

霉菌R.oryzae IFO催化大豆油脂合成生物柴油的研究

直接将霉菌R.oryzae IFO细胞应用于催化大豆油脂转酯合成生物柴油,并对其反应条件进行了优化.在反应所需甲醇分3次等量加入、醇油摩尔比1:1、反应温度35℃、缓冲液用量0.05mL/g油,缓冲液pH值范围3.6～6.98,生物柴油(脂肪酸甲酯)最终得率可达86%.

米曲霉(Aspergillus oryzae FAFU)淀粉酶的分离纯化及其酶学性质研究

从米曲霉（Aspergillus oryzae FAFU）发酵产物中提取淀粉酶,通过硫酸铵盐析、透析、DEAE-Sepharose离子交换层析和Sephacryl S-200凝胶柱层析等纯化步骤,回收活性组分,结合十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行纯度鉴定,共分离纯化出3种酶：酶Ⅰ、酶Ⅱ、酶Ⅲ。通过酶解淀粉实验,酶解产物经薄层层析（TLC）分析,鉴定出酶Ⅰ、酶Ⅱ为液化酶,酶Ⅲ为糖化酶,经SDS-PAGE电泳确定分子量大小分别为：48.6、49.7、93.5 k Da。对酶Ⅰ、酶Ⅱ的酶学性质进行初步的探究：酶Ⅰ的最适反应温度为50℃,温度在50℃以下稳定性好,55℃的半衰期是40 min,最适反应p H为6.0,在p H7.2~9.6范围内稳定性好;酶Ⅱ的最适反应温度为50℃,温度在45℃以下稳定性好,50℃的半衰期是30 min,最适反应p H为6.6,在p H8.6~10.0范围内稳定性好。

固态共培养中丝状真菌对Aspergillus oryzae Su-16的影响

为了研究在固态基质中共培养的两种丝状真菌之间的相互影响,将Aspergillus oryzae Su-16与6株酒曲真菌分别混合接种麸皮培养基进行固态发酵,通过分析麸曲的α-淀粉酶活力、糖化酶活力、中性蛋白酶活力、纤维素酶活力以及麸曲的蛋白质含量、总酸、浸提液的pH值7个指标,借助SPSS16.0软件,利用Spearman相关系数分析,比较共培养麸曲与纯培养麸曲的加合之间的差异性。结合麸曲的SDS-PAGE的图谱分析和十字交叉培养法探讨A.oryzae Su-16在与6株真菌共培养体系中的相互影响的强弱。研究结果表明A.oryzae Su-16在与黑曲霉AN-13共培养中相互影响相对最大,且A.oryzae Su-16明显影响到黑曲霉AN-13的生理代谢而非生长分化,而微小毛霉RP-09则能显著影响到A.oryzae Su-16的分化和产孢。

米曲霉Aspergillus oryzae As-W.6对脱氧雪腐镰刀菌烯醇的降解效果

研究米曲霉Aspergillus oryzae As-W.6对脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)的降解作用。经高效液相色谱(high performance liquid chromatography,HPLC)检测,不同初始质量浓度的DON经菌株As-W.6发酵7 d后,降解率均在35%以上;发酵14 d后,降解率均在90%以上;同时该菌株中提取的降解酶对不同初始质量浓度DON的降解率均达70%以上。对降解产物采用高效液相色谱-质谱(HPLC-mass spectrometry,HPLC-MS)分析检测,结果显示DON经该降解菌降解后,相对分子质量从原来的295.1变为281.2,减少了13.9,说明DON确实被该菌降解成为另一种物质。

Actinomucor elegans、Aspegillus oryzae和Rhizopus oligosporus产蛋白酶条件及蛋白酶性质的比较

比较了腐乳生产菌株Actinomucor elegans、豆酱和酱油生产菌株Aspegillus oryzae以及天培生产菌株Rhizopus oligosporus产生蛋白酶的条件和所产蛋白酶的性质。结果表明,不同的菌株产酶条件及蛋白酶的性质有较大的差异:少孢根霉主要产生酸性蛋白酶,在pH2.5-4.0的酸性介质中、32℃条件下培养时产酶能力较强,所分泌的蛋白酶系在pH5.0时酶活力最高,在pH5.0附近最稳定;米曲霉可以产生酸性、中性及碱性蛋白酶,所产生的蛋白酶活力显著高于少孢根霉和毛霉,米曲霉在酸性条件下产酸性蛋白酶能力强,在中性条件下产中性蛋白酶能力强,在碱性条件下产碱性蛋白酶能力强,在28-32℃时产酶能力强,所分泌的蛋白酶系在pH5.0-9.0的广泛范围内有很强的活力,在pH6.0-8.0的范围内稳定性强;毛霉可以产生酸性、中性及碱性蛋白酶,但酶活力明显低于米曲霉,毛霉在中性偏酸性(pH5.5)的介质中产酸性蛋白酶的能力较强,但介质的酸碱度对毛霉产中性及碱性蛋白酶没有影响,在28℃时产酸性、中性和碱性蛋白酶的能力都比较强,毛霉所分泌的蛋白酶系在pH5.0-9.0的广泛pH范围内有活力,在pH5.0-6.0时酶活力最高,在pH5.0-7.0时稳定强。

水稻对白叶枯病Xanthomonas oryzae pv.Oryzae水平抗性的数量性状位点(QTL)定位

利用“Lemont/特青”重组自交系（RI）群体研究了水稻对白叶枯病致病菌株CR6的水平抗性。双亲和F1均为感病，重组自交系（RILs）的病斑长度（LL）为带有明显双向超亲的连续变异，显示出典型的多基因遗传特征。部分重组自交系（约占总数90％）对CR6表现高水平抗性（LL≤3cm）。利用由178个良好分离的RFLP标记构建的饱和连锁图，鉴定出11个数量形状位点（QTLs）和3对互作位点解释了RI群体的大部分病斑变异。抗性QTLs定位于水稻第2、3、4、8、9、10、11、12等8条染色体。在来自特青的Xa－4位点上检测到一个有很大加性效应的QTL。其余10个QTLs的抗性等位基因有7个来自特青，3个来自Lemont。研究结果表明多个数量性状位点和失效主基因（Xa－4）残效的累加效应构成了对白叶枯病水平抗性的遗传基础，是重要的抗性组成部分。可以预期在DNA标记的辅助下，这些数量性状位点与主效抗性基因的组合将使水稻品种具有持久抗病性。

Aspergillus oryzae SAICAR合成酶基因的克隆及反义表达载体的构建

目的 构建Aspergillus oryzae新的红色表现型菌株用于天然色素生产。方法 构建Aspergillus oryzaeRIB40 cDNA文库,应用BLAST网络服务对Aspergillus oryzae RIB40 cDNA文库的EST数据进行同源性比较,从EST克隆扩增Aspergillus oryzae类SAICAR合成酶基因(sh1919f片段),反向插入pUSA真核表达载体。结果 完成反义表达载体的构建。Aspergillus oryzae类SAICAR合成酶氨基酸序列与Pichia jadinii、Saccharomyces cerevisiae和Schizosac-charomyces pombe的SAICAR合成酶氨基酸序列具有高度同源性。结论 类SAICAR合成酶基因反义表达载体的构建为其转染入Aspergillus oryzae菌株筛选红色突变菌株奠定了实验基础。

RSM法优化Aspergillus oryzae AO-01产糖化酶条件的研究

采用单因素试验研究培养时间、培养温度和固水比对米曲霉(Aspergillus oryzae)AO-01产糖化酶的影响。在此基础上,再采用Box-Behnken响应面法(response surface methodology,RSM)对以上培养条件进行了优化。通过对二次多项回归方程求解得知,在上述自变量分别为培养时间32.8h,培养温度31.2℃,固水比1:1.24时,糖化酶的产量从最低值656.2μ/g干曲提高到1645.1μ/g干曲。验证实验证实了方程的预测值与实际值之间具有较好的拟合度。

水稻品种次生物质与稻白叶枯病(Xanthomonas oryzae pv. oryzae)抗性的关系

应用高效液相色谱分析了对白叶枯病具有不同抗性水平的12个水稻品种中的19个(组)次生物质色谱峰(面积)的差异及其与白叶枯病抗性水平间关系。结果表明,水稻品种抗性水平与谱峰面积值之间相关极显著(R=0.992,p<0.01),被测的19个组分中,峰1、峰2、峰8、峰10、峰12、峰14、峰16和峰18是影响水稻对稻白叶枯病抗性水平的主要抗原次生物质。建立了水稻品种对白叶枯病抗性级别与以上次生物质含量谱峰面积之间的回归模型:Y=10.7603+0.1823X1-0.2287X2+0.2163X8-2.1975X10+0.0728X12-0.7438X14+1.1484X16-0.7795X18。研究结果表明水稻品种中起抗病作用的抗原次生物质不止一种,而是几种的组合,而且它们对水稻抗病性的贡献作用是不完全相同的,这与它们的性质与含量密切相关。提出了以抗原次生物质为标记的快速分析、鉴定、预测水稻品种对稻白叶枯病抗性水平的新途径、新方法。

米曲霉(Aspergillus oryzae)FSO179产α-淀粉酶固体发酵优化的研究

米曲霉(Aspergillus oryzae)FSO179产α-淀粉酶固体发酵优化结果表明:最佳有机碳源为玉米粉,最佳有机氮源为花生饼粉;培养基正交试验表明最佳培养基配方为:花生饼粉20%,玉米粉5%,无机盐为c组配方;初始发酵pH为7.4;最适的发酵温度为33℃;在以上最适条件下固体培养6d,发酵产酶水平可达1300.6u/g,优化结果比初始设计提高了42.9%。该酶酶学特性研究表明:该酶作用的最适温度为55℃;最适作用pH为4.0;Fe2+、Ba2+、Cu2+、Mn2+、Fe3+金属离子对酶具较强抑制作用,而Ca2+对酶具有一定激活作用。