Involvement of OsNPR1/NH1 in rice basal resistance to blast fungus Magnaporthe oryzae

Rice blast disease, caused by the fungus Magnaporthe oryzae, is a major threat to worldwide rice production. Plant basal resistance is activated by virulent pathogens in susceptible host plants. OsNPR1/NH1, a rice homolog of NPR1 that is the key regulator of systemic acquired resistance in Arabidopsis thaliana, was shown to be involved in the resistance of rice to bacterial blight disease caused by Xanthomonas oryzae pv. oryzae and benzothiadiazole (BTH)-induced blast resistance. However, the role of OsNPR1/NH1 in rice basal resistance to blast fungus M. oryzae remains uncertain. In this study, the OsNPR1 gene was isolated and identified from rice cultivar Gui99. Transgenic Gui99 rice plants harbouring OsNPR1-RNAi were generated, and the OsNPR1-RNAi plants were significantly more susceptible to M. oryzae infection. Northern hybridization analysis showed that the expression of pathogenesis-related (PR) genes, such as PR-1a, PBZ1, CHI, GLU, and PAL, was significantly suppressed in the OsNPR1-RNAi plants. Consistently, overexpression of OsNPR1 in rice cultivars Gui99 and TP309 conferred significantly enhanced resistance to M. oryzae and increased expression of the above-mentioned PR genes. These results revealed that OsNPR1 is involved in rice basal resistance to the blast pathogen M. oryzae, thus providing new insights into the role of OsNPR1 in rice disease resistance.     

Effect of carpropamid on secondary infection by rice blast fungus

Carpropamid (WIN™, KTU 3616) provides good control of leaf and panicle blast by ‘one-shot’ nursery-box treatment. It inhibits melanin biosynthesis in appressorial cells of Pyricularia oryzae, making them hyaline. Penetration by infection hyphae from the hyaline appressoria into rice epidermal cells is substantially hindered. In addition, the spread of rice blast spores from primary lesions to the other parts of the plant leading to secondary infection is largely prevented when the plants are treated with carpropamid by spray or water surface application. Secondary infection was simulated in a glass chamber fitted with an ultrasonic humidifier. On treated plants, many blast spores formed in the lesions, but the number of air spora that were dispersed from the lesions decreased significantly. A similar suppression of the spore liberation was observed in vitro when lesions on rice leaf segments, or discs from Pyricularia cultures on oatmeal agar were treated with the chemical. Spores from treated lesions or from the cultures on oatmeal agar amended with the chemical germinated normally and produced well-melanized appressoria on cellophane membranes. In addition, the spores proved to be fully pathogenic towards rice seedlings, producing normal disease symptoms. These results strongly suggest that carpropamid reduces the secondary infection of rice by Pyricularia by specifically hindering spore liberation. © 1999 Society of Chemical Industry     

Effects of panicle development stage and temperature on rice panicle blast infection by Magnaporthe oryzae and visualization of its infection process

Panicle blast, caused by the fungus Magnaporthe oryzae (syn. Pyricularia oryzae), directly contributes to yield loss in the field. The effects of panicle development stage and temperature on panicle blast were studied and the infection process of M. oryzae in panicles was visualized. Rice panicles at different development stages from three rice cultivars were inoculated with a conidial suspension in vitro. The rice cultivar Lijiangxintuanheigu was highly susceptible to panicle blast at 5 days postinoculation (dpi) when the pulvinus distance was 15–20 cm. Nanjing 9108 was moderately susceptible to panicle blast when the pulvinus distance was 8–10 cm, but Yliangyou 800 was resistant. The effect of temperature on panicle blast was determined under 22–35 °C temperature treatments. Inoculated panicles placed at temperatures of 28 and 30 °C showed the highest lesion grade based on lesion length at 5 dpi. The infection process of M. oryzae in rice panicles was observed by confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). M. oryzae initially formed the appressorium to invade through the epidermis of rice panicles at 24 hours postinoculation (hpi). As the disease progressed, the invasive hyphae formed dense mycelial networks in the inner parenchyma cells at 60 hpi. Our results will contribute to the understanding of panicle development stage and temperature effects on panicle blast and improve resistance evaluation methods. Additionally, visualization of the infection process by CLSM and TEM are valuable methods to observe M. oryzae invasive hyphae inside rice panicle cells.     

Resistance in leaf blades assessed by counting conidia correlates with whole-plant-specific resistance in leaf sheaths in a compatible rice–Magnaporthe oryzae interaction

Resistance in the leaf blades of rice plants against a virulent race of the rice blast fungus Magnaporthe oryzae was quantitatively examined using a modified spot inoculation method. Numbers of conidia produced in the lesions were affected by plant age and paralleled the frequency of resistance infection types, which is indicative of whole-plant-specific resistance (WPSR), in the inoculated leaf sheaths of the corresponding plants. Exogenous abscisic acid treatment, which suppresses WPSR, also increased the susceptibility of the leaf blades. These results indicate a correlation between the resistance of the leaf blades and the WPSR in the leaf sheaths.     

Resistance to blast (Magnaporthe grisea) in a mini-core collection of finger millet germplasm

Blast caused by Pyricularia grisea [teleomorph: Magnaporthe grisea] is an economically important and widespread disease of finger millet in the world. Host resistance is the most economical and effective means of combating this disease as finger millet is predominantly grown by resource-poor and marginal farmers. At the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), we evaluated a finger millet mini-core collection of 80 germplasm accessions (about 1 % of the total germplasm collection representing major trait variability) for blast resistance both in the field and greenhouse. Field evaluation was done using a refined screening technique that included new improved rating scales for leaf, neck and finger infection. Sixty six of the 80 accessions showed combined resistance to leaf, neck and finger blast in two seasons (2009 and 2010) of field screening. A highly significant and positive correlation was found between neck and finger blast ratings (r?=?0.92), whereas small but significant correlations were found between leaf blast and neck blast (r?=?0.25) and between leaf blast and finger blast (r?=?0.30). These accessions were also screened for leaf blast resistance in the greenhouse by artificial inoculation of seedlings to confirm field observations. Fifty-eight of the 80 accessions were resistant to leaf blast in the greenhouse screen as well. These resistant accessions represented one wild (africana) and four cultivated races (vulgaris, plana, elongate and compacta) of finger millet that originated from 13 countries in Asia and Africa and exhibited considerable diversity for agronomic traits, such as maturity period, plant height and panicle type. These blast resistant accessions from the mini-core collection would be useful in finger millet disease resistance breeding programs.     

Transgenic rice plants that over-express the mannose-binding rice lectin have enhanced resistance to rice blast

Mannose-binding rice lectin (MRL), which is almost identical to the salt-induced protein SalT, binds to mannose and glucose residues. Expression of the MRL gene in response to infection with Magnaporthe oryzae, the rice blast fungus, was stronger in the incompatible interaction than in the compatible. Transgenic rice plants that constitutively over-expressed MRL strongly suppressed the growth of invasive hyphae of the fungus on leaf sheaths and the development of typical susceptible-type lesions on leaf blades, but did not affect penetration by the fungus in comparison with the wild-type. On a polycarbonate plate, purified recombinant MRL inhibited conidial attachment and appressorium formation but not conidial germination. These results suggest that MRL may play an essential role in disease resistance by suppressing development of M. oryzae in situ.     

OsDCL1基因沉默突变体诱导稻瘟病抗性的转录组分析

稻瘟病是严重危害水稻生长的真菌病害,每年给水稻生产带来重大经济损失。与野生型日本晴NPB相比,OsDCL1基因沉默突变体增强了水稻对稻瘟菌的广谱抗病性。为了解水稻OsDCL1-RNAi突变体的抗病机制,我们分析和比较了它和野生型日本晴NPB在稻瘟菌侵染前后转录组水平的变化。响应稻瘟菌侵染的7 129个差异表达基因(DEGs)中,NPB和OsDCL1-RNAi突变体分别有5 382和5 180个基因表达发生了变化,参与生物胁迫反应、信号通路、蛋白代谢和RNA调控4个通路的基因明显被富集。以野生型未受稻瘟菌侵染的基因表达为对照,在OsDCL1-RNAi突变体中共发现1 318个DEGs,且超过70%是上调的,其中很多基因参与了生物胁迫反应(26.9%)和信号通路(11%),上调表达基因中与生物胁迫反应相关的主要为PR基因和细胞壁相关基因。另外,还发现10个茉莉酸相关基因和11个水杨酸相关基因分别在OsDCL1-RNAi突变体中显著下调和上调表达。在OsDCL1-RNAi突变体中还发现WRKY和MYB等一些转录因子家族以及部分受体类激酶被诱导表达。用qRT-PCR方法验证部分差异表达的基因,其结果与DEGs基本一致。OsDCL1-RNAi突变体中转录组变化的分析,为深入了解该突变体抗瘟性增强的机制奠定了基础。     

枯草芽孢杆菌B-332菌株对稻瘟病的防治效果及定殖作用

为了进一步明确枯草芽孢杆菌B-332菌株对稻瘟病的生防作用,分别对盆栽水稻的防治效果、防治适期、标记菌株在水稻上的定殖作用及田间的防治效果进行了考察。结果表明,在盆栽水稻活体生物测定中,B-332的芽孢菌体悬浮液及发酵上清液对稻瘟病均表现出较好的防治效果(50%以上),且当菌液与上清液混合后其防治效果有加和作用。该菌株对稻瘟病的防治作用主要体现在预防,其预防叶瘟的效果与接种稻瘟病菌时叶面已定殖的B-332菌株的数量有关,接种时水稻叶面上定殖的菌量为1.25×104cfu/mm2时,其防治效果可以达到45%。在人工大量引入的情况下,该菌能够在水稻叶面上有效定殖达21天以上,并且在叶面有10%病斑的情况下,检测到的标记菌株数量会更多。田间试验结果表明,芽孢浓度在1×108cfu/mL时,该菌株对水稻穗颈瘟的防治效果达57.2%,增产率达9.6%,且浓度在5×108cfu/mL时对螃蟹无毒性。     

Use of Myxococcus xanthus protoporphyrinogen oxidase as a selectable marker for transformation of rice

Protoporphyrinogen oxidase (PPO) is the target enzyme of peroxidizing herbicides. The overexpression of Myxococcus xanthus PPO (Mx PPO) confers a high level of herbicide resistance in rice. Among the peroxidizing herbicides, butafenacil has an efficiency ∼1000-fold that of oxadiazon, as judged by calli susceptibility tests upon herbicide treatment. Butafenacil (0.1 μM) was used to select transgenic rice plants expressing Mx PPO under the control of the constitutive maize ubiquitin promoter. The ectopic expression of the Mx PPO transgene was investigated in the T₀ generation by Northern blot and Western blot analysis. The T₀ transgenic plants expressing the Mx PPO gene were resistant to butafenacil based on in vitro leaf disk and in vivo foliar spray tests.     

Expression patterns of defence genes and antioxidant defence responses in a rice variety that is resistant to leaf blast but susceptible to neck blast

The rice stage resistance variety Jiajing3768 is resistant to leaf blast but susceptible to neck blast. This variety was used to analyze the expression patterns of defence genes and antioxidant defence responses in the leaves at the seedling stage (LSS) and necks at the preliminary heading stage (NPHS), after inoculation with Magnaporthe oryzae. We found that defence genes PR1b (pathogenesis-related class 1b), PR4, PR10a, JIOsPR10 (jasmonic acid induced rice PR 10), Gns1 (1,3; 1,4-β-glucanase), Cht-1 (chitinase), and LOX (lipoxygenase) may play roles in the stage resistance in Jiajing3768; PAL (phenylalanine ammonia-lyase) and CHS (chalcone synthase) could participate in defending Jiajing3768 against neck blast but not leaf blast. The antioxidant enzymes superoxide dismutase, peroxidase, catalase, glutathione reductase, and malondialdehyde coordinately participate in the stage resistance to blast in Jiajing3768, and that oxidative damage is less in the LSS than in the NPHS.     

Mining of rice blast resistance gene Pi54 shows effect of single nucleotide polymorphisms on phenotypic expression of the alleles

The rice blast resistance gene Pi54 (formerly Pi-k ^h ) isolated from indica rice line Tetep confers broad spectrum resistance to different strains of Magnaporthe oryzae in India. In this study, we performed PCR based allele mining for blast resistance gene Pi54 from six cultivated rice lines and eight wild rice species to understand its structural variation and its impact on the phenotypes. Sequence analysis indicates presence of more variation between cultivated and wild species (35–90 %) than variation found among cultivated species (1–20 %). Structural analysis of alleles showed presence of variable number of Open Reading Frames (0–2) principally having point mutations in the leucine rich repeats (LRR) regions. The Ka/Ks ratio of LRR region was >1, which shows the effect of selection pressure at this domain. The Pi54 alleles have 142 polymorphic sites with average nucleotide diversity of 0.04522. The K_a/K_s ratio of coding region ranged from 0 to >1 and Tajima’s D test showed negative as well as Darwinian selection within the alleles, which corresponded well with their phenotypic reaction to M. oryzae. The results obtained in this study shows divergent nature of Pi54 allele in wild species and land races of rice. The resistance alleles identified in this study can be used in effective management of rice blast disease through gene pyramiding.     

High activities and mRNA expression of pyrophosphate-fructose-6-phosphate-phosphotransferase and 6-phosphofructokinase are induced as a response to Rhizoctonia solani infection in rice leaf sheaths

Rice sheath blight disease caused by Rhizoctonia solani Kuhn results in significant yield and quality losses in rice growing areas worldwide. The glycolytic pathway is important in the resistance response to R. solani infection in rice. This study examined one of the regulatory steps in this pathway catalyzed by pyrophosphate-fructose-6-phosphate-phosphotransferase (PFP) and 6-phosphofructokinase (PFK). PFP and PFK activity in R. solani-infected rice plants increased. The mRNA expression of PFP/PFK isozymes showed that PFK 1, 2, 4 and 5 in the resistant line at 1 dpi were high as compared to the gradual increase observed in the expression of all PFP isozymes. Also PFK 1, PFK 3, PFK 4, PFK 5, PFP 2 and PFP 5 were adaptive to sheath blight disease infection and linked to defence response while, the expressions of PFK 2, PFP 1, PFP 3 and PFP 4 although adaptive, were not specific to R. solani infection. These observations provide evidence that (a) both PFP and PFK have isozymes that play an adaptive role after R. solani infection but while those of PFK are expressed at higher levels within a short time after infection those of PFP are expressed gradually, (b) the adaptive activation of PFP in R. solani-infected rice plants is correlated with the paired expression of its α- and β-subunits as shown by PFP 2 and PFP 5, and (c) the expression of some α-subunits is not specific to R. solani infection as shown by PFP 1, PFP 3 and PFP 4.     

丙硫磷控制稻瘟病的作用方式及应用技术

经１９９３－１９９５年生物测定和田间多点试验示范发现；１．丙硫磷对稻瘟菌孢子萌发和附着胞形成有显著的抑制活性，ＥＣ５０为１０３．３ｍｈ／Ｌ，５００ｍｇ／Ｌ的抑菌率达９６．２％；２．丙硫磷１０００ｍｇ／Ｌ对水稻苗叶瘟和穗瘟的预防效果分别为７９．４％和９０．７％，治疗效果分别为３９．３％和４１．８％；３丙硫磷６００－９００ｇ／ｈｍ＾２防治双季稻和瓜后稻穗瘟病的田间小区效果分别为７０．８％－８０．２％和     

Leaf blast disease reduction by rice-phyllosphere actinomycetes producing bioactive compounds

Fungal leaf blast, caused by Pyricularia oryzae, is a devastating disease of rice plants that annually causes severe production losses worldwide and is one of the top 10 fungal diseases that threaten global food security. Thus, a reliable control strategy against this disease is essential. In this study, the antagonistic activity of indigenous phyllosphere actinomycetes was elucidated against P. oryzae in vitro and in planta to develop an efficient, effective and environmental friendly approach to protect rice plants against P. oryzae. Of 75 isolates of actinomycetes isolated from the rice phyllosphere, 18 isolates inhibited P. oryzae by >45%. According to analysis of their 16 S rRNA gene sequences, the majority of the 18 isolates belonged to Streptomyces genera; others were identified as belonging to Saccharothrix, Gordonia, or Lentzea. Isolates that potentially produced a bioactive compound(s) were identified among the 18 isolates: 17 isolates (94.44%) had a domain marker for nonribosomal peptide synthetase (NRPS) gene and 12 (66.67%) had type-I polyketide synthase (PKS) gene in their corresponding genome. Interestingly, isolates JSN1.9, SKB2.14, and SKB2.3 suppressed disease suppression by approximately 88%. To our knowledge, this is the first report on the application of rice-phyllosphere actinomycetes producing bioactive compounds to control leaf blast disease in Indonesia. Thus, these findings have escalated the potential application of phyllosphere actinomycetes as a supreme biocontrol agent against fungal leaf blast disease.     

Assessment of blast disease resistance in transgenic PRms rice using a gfp-expressing Magnaporthe oryzae strain

Rice blast caused by the fungus Magnaporthe oryzae (anamorph Pyricularia grisea ) is one of the most devastating diseases of cultivated rice worldwide. In this study, a green fluorescent protein ( gfp )-expressing M. oryzae strain was generated and used to investigate the infection process in a commercial rice cultivar. Expression of the gfp gene did not affect the pathogenicity of the M. oryzae transformants. Confocal microscopy allowed in vivo imaging of this pathogen during infection of rice tissues. Magnaporthe oryzae pathogenicity was examined on both leaf and root tissues. In roots of wild-type plants, the fungus penetrated into epidermal and cortical cells, and colonized the central cylinder and xylem vessels. However, the dimorphic growth pattern typically observed during the biotrophic and necrotrophic stages of leaf colonization was not observed during colonization of root tissues. Furthermore, events occurring during infection of rice plants constitutively expressing the maize pathogenesis-related PRms gene were characterized and compared with those occurring during the interaction of this pathogen with untransformed rice plants. Fungal penetration was drastically reduced and delayed in tissues of PRms plants compared to untransformed plants. These results indicated that the gfp -expressing M. oryzae represents a strategic tool for the assessment of blast disease resistance in transgenic rice which can be also applied to the analysis of the M. oryzae interaction with other cultivars or mutants of important crop species.     

Evaluation of genetic resistance fromOryza sativa andOryza officinalis againstCochliobolus lunatus in Tamil Nadu,India

A new sheath rot disease of rice incited byCochliobolus lunatus (anamorph:Curvularia lunata) was first observed on rice cultivars at the end of the dry season (April and May) in 1990. Nearly 7% of rice plants at boot leaf stage showed typical sheath rot symptoms. Panicles emerging from diseased plants were mostly black. Six improved cultivars and five breeding lines derived from the wild riceOryza officinalis were completely free of the disease under both natural and artificial infection conditions. These genetic stocks with resistance to sheath rot can now be utilized in breeding programs.     

籼型杂交水稻对稻曲病的田间抗性差异

为了解不同水稻品种在田间的抗性差异及其与农艺性状的相互关系,在相同的土壤环境和栽培条件下,利用稻曲病菌诱发接种鉴定,对56个籼型杂交水稻进行田间抗性差异比较,初步探讨了抽穗期和农艺性状与品种田间抗性的关系.结果表明,不同水稻品种对稻曲病田间抗性存在显著差异,病穗率为0.43%～33.04%,病情指数为0.05～16.14;不同抽穗期的品种稻曲病发病率不同,同一抽穗期的品种对稻曲病的抗性亦表现出明显差异;水稻株高与品种抗性之间呈正相关,相关系数为026(P<0.05);水稻分蘖率、有效穗数、剑叶性状等与品种的田间抗性有一定相关性,但差异不显著(P>0.05);水稻株叶形态和叶色与品种田间抗性没有直接相关性.     

云南野生稻和地方稻资源抗白叶枯病分析

为鉴定云南稻种质资源对水稻白叶枯病的抗性情况,于孕穗期采用剪叶接种方法,用水稻白叶枯病强致病型代表菌株BD8438、CN9404和X1接种云南野生稻和地方稻种质资源,以病斑长度大于6 cm为感病分界线,对其抗感表现型进行调查分析。结果显示,共鉴定出来源于云南省不同种植生态区的186份地方稻抗性材料和22个野生稻抗病居群。野生稻对水稻白叶枯病的抗病能力较地方稻强,其中疣粒野生稻的抗性最强,抗病等级为0~2;药用野生稻次之,抗病等级为1~2;普通野生稻相对较差,抗病等级为1~5。地方稻抗性资源来自于云南省各个传统水稻种植区,抗性1级的材料占17%,抗性2级的占2%,抗性3级的占81%;按照稻种质资源亚种类型、粘糯性和水旱性分类,各类型地方稻抗性材料所占比例分别为粳稻占61%、籼稻占39%;粘稻占66%、糯稻占34%;水稻占83%、陆稻占17%。从利用抗白叶枯病基因培育新品种的角度评价,这些抗性资源具有潜在的发掘利用价值。