非亲和性稻瘟病菌对水稻的诱导抗性

为研究稻瘟病菌Magnaporthe oryzae不同菌株间的相互作用,选择与单抗性基因系水稻IRBL5-M （携带抗性基因Pi5）表现为亲和性的菌株HN52与非亲和性的菌株HN119为研究对象,将其单独或混合接种到单抗性基因系水稻IRBL5-M中,并通过荧光显微镜观察接种后水稻叶鞘的发病情况及病斑面积,测定接种后水稻内相关抗性基因OsWRKY45、OsNPR1、OsPR10、OsMAPK2的表达量以及活性氧的变化。结果显示,相较于单独接种亲和性菌株,混合接种后单抗性基因系水稻IRBL5-M病斑发病面积减少;混合接种中亲和性菌株HN52菌丝侵染能力降低,侵染菌丝细胞间扩展率显著降低73.13%;同时单抗性基因系水稻IRBL5-M中OsWRKY45、OsNPR1、OsPR10和OsMAPK2抗性基因表达量显著增加,水稻叶片中活性氧含量增加,表明在菌株混合侵染过程中,非亲和性菌株可通过激发水稻的抗性反应来降低亲和性菌株对水稻的侵染程度。     

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.     

Identification of blast resistance genes in 358 rice germplasms (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) using functional molecular markers

Rice blast, caused by the fungus Magnaporthe oryzae, is one of the most devastating diseases of rice (Oryza sativa L.), and neck blast is the most destructive phase of this disease. Although neck blast causes tremendous yield loss, little is known about the molecular mechanisms underlying the neck blast resistance. To address this issue, we collected 358 rice varieties from different ecotypes in China and assessed them for the neck blast resistance under natural conditions favoring disease development in Jining, Shandong Province. Our results showed that 124 (34.6%) and 234 (65.4%) varieties were resistant and susceptible to M. oryzae under natural field conditions, respectively. Among the 358 rice varieties that were screened for the presence of 13 major blast resistance (R) genes against M. oryzae by using functional markers, 259 varieties contained one to seven R genes. In addition, the relationship between the presence of R genes and the disease reactions was also investigated by integrative analysis of phenotyping and genotyping based on functional markers. Our results showed that the rice blast resistance gene Pi2 was significantly correlated with neck blast resistance. Furthermore, any of the 13 major blast R genes was absent from 32 rice varieties exhibited obvious neck blast resistance, which would be the potential materials for identifying novel neck blast R genes. Taken together, our findings provide insight into the distribution of the 13 major blast R genes in the tested Chinese rice germplasm resources, which will serve as a basis for developing rice blast resistant. Furthermore, 32 rice varieties exhibited neck blast resistance, but they did not harbor any of the 13 major blast R genes. In the future, these varieties may be used to identify novel neck blast R genes.     

Site‐directed mutagenesis of the cytochrome b gene and development of diagnostic methods for identifying QoI resistance of rice blast fungus

BACKGROUND: It is possible that a single nucleotide polymorphism (SNP) (G143A mutation) in the cytochrome b gene could confer resistance to quinone outside inhibiting (QoI) fungicides (strobilurins) in rice blast fungus because this mutation caused a high level of resistance to fungicides such as azoxystrobin in Pyricularia grisea Sacc. and other fungal plant pathogens. The aim of this study was to survey Magnaporthe oryzae B Couch sp. nov. isolates in Japan for resistance to QoIs, and to try to develop molecular detection methods for QoI resistance. RESULTS: A survey on the QoI resistance among M. oryzae isolates from rice was conducted in Japan. A total of 813 single‐spore isolates of M. oryzae were tested for their sensitivity to azoxystrobin using a mycelial growth test on PDA. QoI fungicide resistance was not found among these isolates. The introduction of G143A mutation into a plasmid containing the cytochrome b gene sequence of rice blast fungus was achieved by site‐directed mutagenesis. Molecular diagnostic methods were developed for identifying QoI resistance in rice blast fungus using the plasmid construct. CONCLUSION: As the management of rice blast disease is often dependent on chemicals, the rational design of control programmes requires a proper understanding of the fungicide resistance phenomenon in field populations of the pathogen. Mutation of the cytochrome b gene of rice blast fungus would be specifically detected from diseased leaves and seeds using the molecular methods developed in this study. Copyright © 2009 Society of Chemical Industry     

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.     

Genetic distribution of the avirulence gene AVRPiz-t in Thai rice blast isolates and their pathogenicity to the broad-spectrum resistant rice variety Toride 1

Rice blast disease, caused by the filamentous fungus Pyricularia oryzae, is one of the most destructive diseases in rice worldwide. Breeding of resistant rice cultivars remains a cost-effective and environment-friendly means for controlling blast disease, but the resistance tends to break down over time because of the pathogen's rapid adaptation. In this study, AVRPiz-t gene sequences of 46 rice blast isolates were evaluated using a Southern blot analysis. The AVRPiz-t gene was present in 24 of 46 (52.2%) rice blast isolates. The pathogenicity assay showed that all blast isolates were avirulent against Japanese rice cv. Toride 1, which carries several rice blast resistance genes including Piz-t, Pii, Pi37, and Pi-ta. Screening for the Piz-t gene in Thai rice germplasm revealed that less than 20% of rice varieties harbour the Piz-t gene. Therefore, the Toride 1 rice variety could serve as an effective donor of rice blast resistance to be used in rice breeding programmes in Thailand. This study provides evidence for co-evolution between the rice blast resistance gene Piz-t and the rice blast fungal avirulence gene AVRPiz-t. Understanding this relationship will facilitate the sustainable development of breeding for rice blast resistance in the future.     

Antifungal activity of a novel compound purified from the culture filtrate of <Emphasis Type="Italic">Biscogniauxia</Emphasis> sp. O821 against the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Rice blast is a devastating fungal disease resulting in major losses to rice crops. Owing to continuous acquisition of resistance by the causal fungus, several fungicide chemicals are no longer effective. Therefore, there is a need to identify natural components and develop new agents to control fungal pathogens. We previously demonstrated that the culture filtrate of Biscogniauxia sp. O821 inhibited infection behavior of Magnaporthe oryzae and subsequent blast lesion formation. In the present study, we isolated a new compound, (3aS,4aR,8aS,9aR)-3a-hydroxy-8a-methyl-3,5-dimethylenedecahydronaphto[2,3-b]furan-2(3H)-one (HDFO), from the culture filtrate of Biscogniauxia sp. O821 and determined its molecular weight as 248. The HDFO structure was determined by electrospray ionization-mass spectrometry and nuclear magnetic resonance spectroscopy after purification with column chromatography and high-performance liquid chromatography. The structure of this antifungal compound was similar to that of alantolactone and isoalantolactone. The growth inhibition zone against M. oryzae in presence of HDFO was observed at Rf 0.5–0.6 on a thin layer chromatography plate. HDFO inhibited conidial germination of M. oryzae in a dose-dependent manner (1–200 ppm). Furthermore, blast lesion formation was significantly suppressed by HDFO at over 5 ppm. These results suggest that HDFO from the culture filtrate of Biscogniauxia sp. O821 can protect rice from rice blast disease caused by M. oryzae. This is the first report that HDFO produced by Biscogniauxia sp. can serve as an antifungal compound against M. oryzae.     

乙烯信号传导途径因子OsEIL 6调控水稻抗稻瘟病反应

稻瘟病(rice blast)是水稻生产上最严重的病害之一。抗病相关基因的挖掘对稻瘟病的防治具有重要意义。研究表明植物EIN3/EIL家族基因在抗病过程中发挥着重要作用。本研究采用RNAi技术探究OsEIL6参与的水稻抗稻瘟病反应。稻瘟菌侵染时基因表达谱检测结果表明,OsEIL6在水稻和稻瘟菌非亲和组合中受到诱导表达。稻瘟菌接种结果显示,水稻OsEIL6沉默株系和野生型植株‘TG394’相比抗性下降;实时荧光定量RT-PCR结果分析表明,OsEIL6的表达量下降导致乙烯合成途径中OsACO1和乙烯信号传导途径的OsERF063和OsERF073的转录水平下降。亚细胞定位研究发现该基因定位于水稻细胞质。OsEIL6沉默株系中ROS合成途径标记基因OsrbohA和OsrbohB的表达量均明显下调,表明该基因可能通过影响ROS的合成调控水稻抗稻瘟病反应。本研究结果将有助于进一步揭示OsEIL6参与的乙烯信号传导途径介导的水稻抗稻瘟病反应机制。     

黑龙江省水稻种质抗瘟性及稻瘟病菌致病性分析

为明确黑龙江省水稻种质抗性及稻瘟病菌的致病性,以黑龙江省8个水稻品种、24个单基因系作为供试材料,120株稻瘟病菌株作为接种体,采用喷雾接种法测定了各供试水稻的抗瘟性及稻瘟病菌的致病性。结果表明,水稻品种对2010年和2011年菌株的抗性频率分别在31.67%~68.33%和21.67%~55.00%之间,2010年最好的抗性品种为松粳12,2011年最好的抗性品种为五优稻4和东农425;松粳12东农425组合联合抗病性最好。水稻单基因系对2010年和2011年菌株的抗性频率分别在10.00%~90.00%和5.00%~86.67%之间,抗性最好的单基因系分别为IRBLzt-T(Pi-zt)和IRBLz5-CA(Pi-z5);松粳12、东农425和龙粳22的基因聚合效果最好。2010年和2011年菌株对抗瘟基因群的致病率分别在8.33%~95.83%和25.00%~95.83%之间;无毒基因总出现频率分别为461和412次。研究表明,水稻种质抗性受菌株致病性影响较大,但高抗种质相对稳定,基因聚合方式更适宜当地品种抗性改良。     

Overwintering of <Emphasis Type="Italic">Pyricularia oryzae</Emphasis> in wild infected foxtails

The rice blast fungus Pyricularia oryzae mainly overwinters in infested rice organs stored indoors, whereas it is difficult or impossible for the pathogen to overwinter outdoors. By contrast, blast pathogens infecting weed grasses must overwinter outdoors every winter to continue their life cycle. In this study, we investigated the overwintering location of P. oryzae infecting wild, green, and giant foxtails to identify the mechanism that enables them to overwinter. Recovery of P. oryzae was tested in seeds of wild foxtail collected from the soil surface from December to April over three winters. No P. oryzae was recovered from the seed samples of any wild foxtail collected at the ends of the three experimental periods in April. Recovery was also tested from blast lesions on leaves and seeds sampled from withered green foxtail in the experimental field of Saga University from November to April during two winters. In contrast to seeds on the soil surface, P. oryzae survived in lesions and seeds at the ends of the two experimental periods during April, suggesting that withered host plants could be the overwintering site of the pathogen. Rice plants are reaped and removed from paddy fields after harvesting. Thus, withered, standing plants may be available solely to blast pathogens infecting wild grasses, possibly explaining the higher winter survival frequency of weed pathogens than that of rice blast pathogens outdoors.     

Suppression of rice blast using freeze-killed mycelia of biocontrol fungal candidate MKP5111B

Several applications of the suppressive fungus MKP5111B, isolated from the phylloplane of rice plants, were tested in an effort to control rice blast disease. Three treatments with MKP5111B [living (Std), killed with liquid nitrogen (FR), and autoclaved (AC)] were either sprayed onto rice seedlings or mixed into seed-sown soil. Three weeks after spraying and 4 weeks after the soil application, we introduced Magnaporthe oryzae, the causal agent of rice blast, into our systems. The Std and FR treatments suppressed rice blast, but the AC treatment proved ineffective. Although a suppressive effect was seen on new leaves, no mycelium of MKP5111B was seen. The fungus thus may have induced a systemic resistance in the rice plants. A substance from MKP5111B, such as elicitor molecule(s) are likely responsible for the induced resistance.     

Infection by Magnaporthe oryzae chrysovirus 1 strain A triggers reduced virulence and pathogenic race conversion of its host fungus, <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Magnaporthe oryzae chrysovirus 1 strain A (MoCV1-A) is associated with an impaired growth phenotype of its host fungus, Magnaporthe oryzae. In this report, we assayed the virulence and pathogenicity of MoCV1-A-infected and MoCV1-A-free M. oryzae on rice plants. MoCV1-A infection did not affect virulence-associated fungal traits, such as conidial germination and appressorium formation. However, after punch inoculation of leaves on rice plants, MoCV1-A-infected strain formed smaller lesions than the MoCV1-A-free strain did on all rice varieties tested, showing that MoCV1-A infection resulted in reduced virulence of host fungi in rice plants. In contrast, after spray inoculation of rice seedlings, in some cases, MoCV1-A-infected and MoCV1-A-free strains caused different lesion types (resistance to susceptible, or vice versa) on individual international differential rice varieties. However, we did not find any gain/loss of the fungal avirulence genes by PCR, suggesting that MoCV1-A infection can convert the pathogenicity of the host M. oryzae from avirulence to virulence, or from virulence to avirulence, depending on the rice variety. We also confirmed the correlation of these race conversion events and invasive hyphae growth of the fungi in a leaf sheath inoculation assay. These data suggested that MoCV1-A infection generally confers hypovirulence to the fungal host and could be a driving force to generate physiological diversity, including pathogenic races.     

Races of <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> in Australia and genes with resistance to these races revealed through host resistance screening in monogenic lines of <Emphasis Type="Italic">Oryza sativa</Emphasis>

Rice blast is the most serious disease threat to rice production worldwide. It is difficult to control due to the complex diversity and wide geographic distribution of the causal pathogen Magnaporthe oryzae. In Australia, rice blast occurs in northern Australia but remains exotic to the main south-eastern rice growing area; however, there is the potential for rice blast to threaten this area; in addition, rice production is currently expanding from south-eastern Australia into northern Australia, which makes rice blast a major concern and challenge to rice industry in Australia. Prior to this study, there was lack of information on the race status of M. oryzae present in Australia and on how to manage the disease through host resistance. The races of rice blast isolates collected in northern Australia was characterised based on the disease reactions of eight standard rice differentials used in an international race differential system. The following studies revealed genes conferring resistance to these races through investigating the responses of 25 monogenic rice lines with targeted resistance gene against different races. The rice blast isolates were characterised into five races: IA-1, IA-3, IA-63, IB-3 and IB-59. Genes Pi40, Piz-t, Pi9, Pi5(t) and Pi12(t) exhibited resistance to all the isolates belonging to five races. In addition, two genes showed complete resistance to multiple races, viz. Pi9 that showed complete resistance to races IA-1, IA-3, IA-63 and IB-3 and Pita2 that had complete resistance to races IA-3, IB-3 and IB-59. This study provides information about the races of M. oryzae in Australia. Genes identified conferring resistance to multiple races will not only streamline the identification via molecular markers of imported rice varieties with resistance to rice blast in Australia, but will also allow the Australian rice breeding program to develop new varieties with broad-spectrum resistance to rice blast and pyramid multi-gene resistance into Australian rice varieties.     

Antagonistic activity against rice blast disease and elicitation of host-defence response capability of an endophytic Streptomyces albidoflavus OsiLf-2

Rice blast, caused by the fungus Magnaporthe oryzae, can result in notable yield losses in rice production. The objective of this study was to investigate the potential of a rice endophytic isolate, Streptomyces albidoflavus OsiLf-2, on the control of rice blast and the possible mechanisms involved. In vitro assays displayed a variety of antagonistic effects of OsiLf-2 against different physiological races of M. oryzae, with peak mycelial growth inhibition ranging from 74.1% to 83.0%. In vivo tests of OsiLf-2 showed 18.0% and 19.6% reduction in disease index in greenhouse and field conditions, respectively. The stable active metabolites in its cell-free culture filtrate inhibited the mycelial growth, spore germination and appressorial formation of M. oryzae in a dose-dependent manner. They also possessed strong antifungal capacities toward various phytopathogens in vitro. OsiLf-2 secreted multiple antimicrobial compounds, cell wall degradation enzymes, siderophore, plant hormones, and 1-aminocyclopropane-1-carboxylate deaminase, which might function in direct or indirect resistance to M. oryzae. In addition, a variety of defence responses were induced in OsiLf-2-treated rice, including hydrogen peroxide (H₂O₂) accumulation, callose deposition, defence-related enzymes activation, and elevated expression of salicylic acid (SA) and jasmonic acid (JA) pathways genes, which might contribute in resisting pathogen attack. The significant biological control activity and host defence-stimulation ability of OsiLf-2 suggest that this endophytic actinobacterial strain could be a promising candidate in the management of rice blast disease.     

Long‐term survival of blast pathogen in infected rice residues as major source of primary inoculum in high altitude upland ecology

Magnaporthe oryzae is the fungal plant pathogen that causes rice blast. The sources of primary inoculum and overwintering mode of the fungus remain largely unknown. The effect of rice residues on the onset of blast epidemics and the potential for survival of M. oryzae in the residues were studied in upland conditions in Madagascar. Blast disease was observed in a 3‐year field experiment in three treatments: with either infected or uninfected rice residues on the soil surface, or without rice residues. Leaf blast incidence was significantly higher in the treatment with infected rice residues than in the two other treatments at the early stages of the epidemic. In a second set of trials, the survival of M. oryzae on rice residues was monitored. Infected rice stems were placed by lots in three places: on the mulch of rice residues, under the mulch, and buried at a depth of 10 cm in the soil. Each month, samples were taken from the field and tested for sporulation. The survival of the blast fungus decreased rapidly on the stems buried in the soil but remained high for the other conditions. Sporulation of the fungus was observed on stems left on the mulch for up to 18 months. It is concluded that under field conditions, the presence of infected rice residues could initiate an epidemic of blast. The results of this study may help in designing effective management strategies for rice residues infected by M. oryzae.     

Green-odour compounds have antifungal activity against the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Four green-odour compounds—trans-2-hexenal, cis-3-hexenol, n-hexanal, and cis-3-hexenal—were applied (0.85 μg ml⁻¹ as vapour) to rice plants in laboratory conditions to observe their biological activity against the phytopathogenic fungus Maganporthe oryzae, which causes rice blast disease worldwide. Two compounds, trans-2-hexenal and cis-3-hexenal, showed remarkable disease suppression efficacy (99.7% and 100% suppression, respectively), while n-hexanal had moderate (86.5%) and cis-3-hexenol had weak (20.8%) disease-suppressing effects. Pre-application and post-application of trans-2-hexenal or cis-3-hexenal had slight effects on blast incidence, suggesting that these compounds had direct effects to suppress M. oryzae infection. In fact, trans-2-hexenal and cis-3-hexenal exhibited a growth suppression effect on M. oryzae. Interestingly, these two compounds inhibited appressorium formation at lower concentrations than the growth suppression. Studies on the hypersensitive response (HR)-like reaction and plant β-1,3-glucanase activity in rice plant confirmed that induced resistance was not the major factor involved in the disease suppression mechanism. Results of this study conclusively showed that trans-2-hexenal and cis-3-hexenal possess potent inhibitory activities against the growth and the appressorium formation of M. oryzae and could be used as antifungal agents to significantly reduce M. oryzae infections in rice.     

In vitro and in vivo antifungal activities of decursin and decursinol angelate isolated from Angelica gigas against Magnaporthe oryzae, the causal agent of rice blast

Blast is considered the most important fungal disease of rice due to its wide distribution and destructiveness under favorable conditions. Development of new effective and environmentally benign agents against the causal pathogen, Magnaporthe oryzae, is of great interest. In the course of a search for natural antifungal compounds in medicinal plants, we found that the methanol extract of Angelica gigas roots showed a potent control efficacy against rice blast caused by M. oryzae. We isolated antifungal coumarins from the extract, and they were identified as decursin and decursinol angelate. Antifungal activities of these compounds, along with kasugamycin, were tested on M. oryzae in vivo and in vitro. In an in vivo assay, the three compounds effectively suppressed the development of rice blast at concentrations more than 100 μg/mL. Coumarins showed relatively weak inhibitory effect on fungal mycelial growth when compared to kasugamycin. However, they strongly inhibited M. oryzae spore germination, which was not observed in kasugamycin treatments. This is the first report demonstrating that decursinol angelate can provide control against rice blast and that the two coumarins inhibit M. oryzae spore germination. In addition, the wettable powder formulation of the crude extract of A. gigas prohibited the development of blast symptoms on rice plants more effectively than liquid concentrate formulation of kasugamin, a commercial fungicide. Based on our study, we propose that coumarin compounds as well as the A. gigas root crude extract can be used as natural, benign fungicides for controlling rice blast.     

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.