Vertical distribution of leaf blast lesions in mixtures of rice cultivar Sasanishiki and its resistant near-isogenic line

The vertical distribution of leaf blast lesions caused by the fungus Pyricularia grisea was studied to estimate the degree of leaf blast suppression in rice multilines in experimental paddy fields for 4 years. Leaf blast in 1 : 1 and 1 : 3 mixtures of susceptible rice cultivar Sasanishiki and its resistant near-isogenic line, Sasanishiki BL7, developed slower than that in pure stands of Sasanishiki. The average distance of lesions on leaves from the ground in the 1 : 3 mixtures was significantly lower than that in the pure stands at the end of leaf blast epidemics (at booting stage). This result shows that the distribution of leaf blast lesions in the upper layer differs between the susceptible pure stands and the 1 : 3 mixtures at the end of leaf blast epidemics.     

Silicon content in rice seedlings to protect rice blast fungus at the nursery stage

To control rice blast effectively at the nursery stage, the absolute SiO₂ content necessary for rice plants to resist blast disease was investigated using various rice cultivars and soils. Nine rice cultivars with different complete resistance genes and different degrees of partial resistance were grown on nursery soils amended with silica gel at different rates to change the SiO₂ content of rice plant. The rice seedlings were then inoculated 28 days after sowing with Pyricularia grisea to estimate their blast resistance. In all rice cultivars, the number of lesions was significantly reduced when SiO₂ content increased in the rice seedling; lesions were reduced to 5%–20% of the number on the seedlings grown in soil without silica gel when the seedling SiO₂ content reached 5%. Additionally, the susceptibility to blast disease of rice seedlings grown on eight soils collected from different districts, with varying amounts of silica gel, was compared. The number of lesions decreased significantly when the SiO₂ content in the seedlings reached 5%. These results suggest that SiO₂ content of at least 5% in the rice plant can control this disease at the nursery stage under any conditions.     

Suppression of rice blast by phylloplane fungi isolated from rice plants

Rice phylloplane fungi were evaluated for their potential as biocontrol agents for rice blast disease caused by Magnaporthe grisea. A total of 1923 fungal isolates were obtained from rice plants in fields at Ishigaki and Iwama and from potted plants placed in a cedar woods in Iwama as bait. Although 82.9% of isolated fungi could not be identified, species of Epicoccum were the most prevalent among identified isolates. Of the 1923 isolates, 967 were randomly selected for screening against rice leaf blast. Nine isolates (MKP5111B, MKP5112, J2JMR3-2, K2J131-2, I5R3-1, NOP541, K1KM134-1, NOP5112, MKP33222) suppressed the disease when a conidial or hyphal suspension of both the phylloplane fungus and pathogen were simultaneously used to inoculate rice plants cultured in pots in a growth chamber. Five of the isolates originated from potted plants in the woods and four from Ishigaki, a subtropical island. Five (MKP5111B, MKP5112, NOP541, NOP5112, MKP33222) of the nine isolates strongly suppressed conidial germination of M. grisea (0.7%) and formed inhibition zones (3–5mm width) in dual cultures with the pathogen. Methanol extracts from the isolates also inhibited mycelial growth of the pathogen. These results suggest that the five isolates produced antibiotic(s). These five isolates are likely identical or closely related fungal species because the sequence of their ITS regions were 100% similar. ITS sequence analysis also suggested that J2JMR3-2 was associated with a species of Fusarium. Under field conditions, J2JMR3-2 reduced both leaf and panicle blast severity, and three other isolates (MKP5111B, K1KM134-1, K2J131-2) suppressed leaf blast in one of the three experiments.     

Effects of preinoculation with an avirulent isolate of Pyricularia grisea on infection and development of leaf blast lesions caused by virulent isolates on near-isogenic lines of Sasanishiki rice

Eight near-isogenic Sasanishiki rice lines with different genes for complete resistance to rice blast were inoculated with an avirulent isolate 72 h before inoculation with a virulent isolate of Pyricularia grisea to clarify the mechanisms of induced resistance in the leaf blades. Subsequent lesions on the leaf blades were classified as brown spots (b type), as observed on Sasanishiki BL no. 8 with resistance gene Pii, or no symptoms (HR type), as observed on Sasanishiki BL no. 4 with the gene Piz-t and on the six other lines. Lesion expansion was significantly reduced in Sasanishiki BL no. 8 compared with that in Sasanishiki BL no. 4 when the leaf blades were preinoculated with a high concentration of a conidial suspension of the avirulent isolate. Moreover, after preinoculation with the conidial suspension of the avirulent isolate in silicon rings on the leaf blades, induced resistance was expressed only in areas close to the inoculation sites. These resistant areas were larger in Sasanishiki BL no. 8 than in BL no. 4. Hyphal growth was markedly inhibited in the epidermal cells of Sasanishiki BL no. 8, whereas inhibition was weak in those of Sasanishiki BL no. 4. In the epidermal cells of leaf blades of Sasanishiki BL no. 8 preinoculated with the avirulent isolate, the frequency of hyphal penetration of the virulent isolate in the presence of host cell browning decreased, as did the frequency of invading hyphae after inoculation with virulent isolates. The results indicate that induced resistance may play a role in the suppression of lesion development in the Sasanishiki near-isogenic lines and that the lines differ in the extent of suppression.     

Effects of leaf blast on growth and production of a rice crop

Rice crops grown under irrigated conditions were inoculated withPyricularia oryzae during early growth stages to study the effect of leaf blast on yield formation. The inoculations led to severe epidemics of leaf blast around maximum tillering, characterized by the presence of typical blast lesions and an accelerated senescence of heavily infested leaf tissue. Leaf blast led to a prolonged tillering and a delay in flowering and maturity.Crop growth rate and leaf area formation declined sharply during establishment of the disease and continued to be reduced till maturity. This resulted in a marked reduction of total dry matter production and grain yield. Dry matter distribution was not affected. Leaf blast reduced spikelet number, 1000 grain weight, and the fraction filled grains. From this last observation it was concluded that the reduction in grain yield was exclusively source determined.Nitrogen uptake of the inoculated crops before flowering was reduced compared to the N uptake of the control crop, but shoot N content of the inoculated crops at flowering was higher. Uptake of nitrogen after flowering was negligible in both healthy and inoculated crops. Redistribution from vegetative tissue was therefore the main source of N for grain growth. The higher N content of the shoot organs in the inoculated crops during ripening led to the conclusion that the reduced N uptake was not responsible for the yield reduction observed. Consequently, the reduction in grain yield was solely determined by a reduced carbohydrate supply.     

Effects of leaf blast on photosynthesis of rice. 1. Leaf photosynthesis

The effect ofPyricularia oryzae, the causal organism of leaf blast in rice, on photosynthesis characteristics of rice leaves was measured in two greenhouse experiments. Leaf blast reduced photosynthesis not only through a reduction in green leaf area, but also through an effect on photosynthesis of green leaf tissue surrounding the lesions. The assimilation rate at light saturation (P _max ) was more affected than the initial light use efficiency (). Dark respiration (R _d ) increased as a result of infection. The experimental data were used to derive relations between leaf blast severity andP _max , andR _d .     

Effects of leaf blast on photosynthesis of rice 2. Canopy photosynthesis

The effect ofPyricularia oryzae, the causal organism of leaf blast in rice, on photosynthetic rate of a rice crop was determined with mobile equipment in the field. Canopy CO₂ exchanges rate (CER) was significantly reduced in plots inoculated withP. oryzae. The experimentally obtained data were used to evaluate the performance of a model for the effects of leaf blast on canopy photosynthesis. The model comprised photosynthesis and respiration routines of a mechanistic crop growth model, extended with submodels for effects of leaf blast on both processes. Canopy photosynthesis and the effect of leaf blast on CER were accurately simulated with the model.Analysis showed that the reduction in canopy photosynthesis was mainly due to an adverse effect of lesions on leaf photosynthetic rate and to shading by dead leaf are resulting from disease induced senescence. A sensitivity analysis demonstrated the importance of the vertical distribution of the disease in the canopy. This implies that disease monitoring for crop loss assessment should consider vertical disease distribution.     

Molecular and Pathotype Analysis of the Rice Blast Fungus in North Vietnam

Rice blast caused by Pyricularia oryzae is a devastating disease worldwide. In Vietnam, rice blast is especially severe in the Red River Delta in the North. The genetic diversity of 114 P. oryzae isolates collected from rice in 2001 in the Red River Delta and nine additional Vietnamese P. oryzae isolates was analysed using Amplified Fragment Length Polymorphism (AFLP). DNA similarity and cluster analysis based on 160 polymorphic AFLP markers showed twelve different AFLP genetic groups among the 123 field isolates. Isolates collected from japonica hosts clustered separately from indica host isolates with at least 60% dissimilarity with little evidence for gene flow between the two populations. In the 2001 population originating from indica hosts, three genetic groups were predominant and represented 99% of the isolates sampled. One predominant clonal lineage represented 59% of the 2001 indica host population and was found in eleven provinces in the Red River Delta of North Vietnam. Significant genotype flow could be demonstrated between the indica population south of Red river and the indica population north of Red river. There was significant linkage disequilibrium between the AFLP loci within the indica population, indicating that this is not a random mating population. Pathogenicity tests of 25 isolates selected from the 12 AFLP groups on a set of 29 differential rice lines revealed two avirulent isolates and 23 pathotypes. Different combinations of known resistance genes were found to have potential for blast resistance breeding for North Vietnam. First two authors contributed equally     

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.     

Morphological studies on attachment of spores of <Emphasis Type="Italic">Magnaporthe grisea</Emphasis> to the leaf surface of rice

Rice leaves were inoculated with spores of Magnaporthe grisea, and the number of fluorescence-labeled spores that attached to the leaf surface were counted before and after leaves were dipped and then stirred in water. Just 5% of the spores were retained on the leaf surface 1h after inoculation; the percentage retained then increased rapidly between 1.25 and 1.50h, and most had attached by 2h. Scanning electron microscopy revealed that most conidia were lying on a few wart-like protuberances 2–4µm high. Spores became attached when the germ tubes were long enough to reach the leaf surface, at least 3µm, by mucilaginous substances at the tip. Retained spores swayed when water was added under the cover glass from one side, indicating that the attachment was confined to the tips of germ tubes. Spores are attached to the rough leaf surface by mucilaginous substances – not at the tip of spore as reported on smooth artificial substrates but at the tip of the germ tubes.     

Effect of glyphosate on tryptamine production and Sekiguchi lesion formation in rice infected with Magnaporthe grisea

When exposed to light, the Sekiguchi lesion (sl) rice mutant has an enhanced resistance to Magnaporthe grisea infection responsible for Sekiguchi lesion formation and tryptamine accumulation. Glyphosate [N-(phosphonomethyl) glycine] pretreatment suppressed Sekiguchi lesion formation and tryptamine accumulation in the sl mutant after M. grisea infection even under light. This inhibition by glyphosate was blocked by the supply of exogenous tryptophan, but not by exogenous phenylalanine. In glyphosate-pretreated leaves, 5-enol-pyruvyl-shikimate-3-phosphate synthase gene expression and tryptophan biosynthesis were significantly suppressed. During tryptophan starvation, catalase activity was maintained at a high level even under light, leading to the suppression of H₂O₂ generation and DNA fragmentation. These results show a strong relationship between the tryptophan and tryptamine pathways in the induction of light-enhanced resistance to M. grisea infection in the sl mutant.     

Oxidative stress induces high transcription of Rhm52 and Rhm54, novel genes identified as being involved in recombinational repair in Magnaporthe grisea

The first two recombinational repair genes of Magnaporthe grisea were cloned. Analysis of the deduced amino acid sequences revealed that Rhm52 and Rhm54 are Saccharomyces cerevisiae RAD52 and RAD54 homologs, respectively. Phenotypic complementation testing of these genes showed their function in recombinational repair. Both genes were in single copies in M. grisea genome. Expression of these genes was induced by methyl methanesulfonate and ultraviolet radiation as known for other homologs of the RAD52 epistasis group. Higher induction of both genes by oxidative stress and heat shock indicated the probability for recombinational repair during the infection cycle of M. grisea. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB091332 and AB091330     

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.     

Identification and mapping of a rice blast resistance gene Pi-g(t) in the cultivar Guangchangzhan

A single dominant blast resistance gene was identified in Chinese indica rice ( Oryza sativa ) cv. Guangchangzhan (GCZ), which shows complete resistance to Japanese isolate Ken53-33 of Magnaporthe grisea . Genetic analysis of the backcross (BC₁) and second-generation (F₂) populations from a cross between susceptible cv. Lijiangxintuanheigu (LTH) and GCZ indicated that the resistance was conferred by one dominant gene. This gene was mapped on the long arm of chromosome 2 and flanked by RM166 and RM208 at distances of 4·00 ± 4·90 and 6·30 ± 4·89 cM (centiMorgans), respectively. It was designated tentatively as Pi-g(t) .     

Occurrence of an antifungal principle in the root extract of a Bayoud — resistant date palm cultivar

An hexane extract from roots of Black Boo Stammi, a cultivar of date palm, resistant toFusarium oxysporum f. sp.albedinis, contained substances which inhibited the spore germination and the growth of the germ tubes of the three isolates of the pathogen that were tested. Extract from roots of Jihel, a susceptible cultivar, lacked these substances.Samenvatting In een hexaanextract van wortels van de dadelpalm, cv. Black Boo Stammi die resistent is tegen de Bayoudziekte, bevonden zich stoffen die de sporekieming en de groei van kiembuizen onderdrukken. Dit gold voor alle drie isolaten vanFusarium oxysporum f. sp.albedinis, die werden getoetst. In extracten van de vatbare cultivar Jihel werden de kieming en de groei niet geremd.     

DNA fingerprinting of Pyricularia grisea by rep-PCR using a single primer based on the terminal inverted repeat from either of the transposable elements Pot2 and MGR586

We investigated the use of single primers complementary to sequences in the terminal inverted repeat (TIR) of either Pot2 or MGR586, transposable elements found in Pyricularia grisea, for DNA fingerprinting by repetitive-element-based polymerase chain reaction (rep-PCR). Under standard amplification conditions, rep-PCR with each single primer generated distinct fingerprint patterns among rice-infecting P. grisea isolates collected in Japan. With the Pot2-TIR primer, bands ranging in size from 0.2 to 8 kb and in number from 8 to 13 per isolate were amplified. Although fewer bands were amplified with the MGR586-TIR primer, this molecular technique should be more reliable to identify and classify P. grisea isolates by combining the data of fingerprint patterns from each TIR primer. In a cluster analysis based on DNA fingerprints from this rep-PCR with the Pot2-TIR primer, 10 reference isolates and 12 field isolates from Saga Prefecture in 2002 were separated into six clonal lineages. We also demonstrated that the 12 field isolates belonged to one clonal lineage. Thus, this rep-PCR method using the single primer Pot2-TIR will be useful for the analysis of the population structure of rice blast pathogens.     

Decrease by plant development and leaf age of susceptibility of groundnut to rust (Puccinia arachidis) in a susceptible cultivar

The effects of plant development and leaf age on the infection efficiency (IE), the latency period (LP) and the sporulation intensity (SP) of groundnut rust were studied using detached and attached leaflets of a highly susceptible groundnut cultivar. The results indicate a decrease ofIE with increasing leaf age and an increase ofLP with increasing leaf age and development stage. A significant effect of detachment onIE was found. However, experiments on both detached and non-detached leaflets resulted in the same, general conclusions. The observed reduction ofIE and lengthening ofLP suggest that further studies would profitably distinguish epidemiologically different layers in the host canopy.Samenvatting De invloed van het ontwikkelingsstadium van de plant en van de leeftijd van het blad op de infectie-efficiëntie (IE), de latentieperiode (LP) en de sporulatie-intensiteit (SP) van aardnootroest werd onderzocht bij een zeer vatbare aardnoot-cultivar aan wel en niet afgesneden deelblaadjes. De resultaten laten een afname zien vanIE bij toenemende bladleeftijd alsmede een toename vanLP met de toename van bladleeftijd en ontwikkelingsstadium. Het effect van het afsnijden van de deelblaadjes opIE was significant, maar proeven met wel en met niet afgesneden blaadjes leidden tot dezelfde algemene gevolgtrekkingen. De waargenomen afname vanIE en verlenging vanLP doen vermoeden dat voortgezet onderzoek een nuttig onderscheid zal kunnen maken tussen in epidemiologische zin verschillende bladlagen van het gewas.     

Investigation of rice blast development in susceptible and resistant rice cultivars using a gfp‐expressing Magnaporthe oryzae isolate

In this study, an isolate of Magnaporthe oryzae expressing the green fluorescent protein gene (gfp) was used to monitor early events in the interaction of M. oryzae with resistant rice cultivars harbouring a blast resistance (R) gene. In the resistant cultivars Saber and TeQing (Pib gene), M. oryzae spores germinated normally on the leaf surface but produced morphologically abnormal germ tubes. Germling growth and development were markedly and adversely affected in leaves of these resistant cultivars. Penetration of host cells was never seen, supporting the idea that disruption of germling development on the leaf surface might be one of the resistance mechanisms associated with Pib function. Thus, this particular R gene appeared to function in the absence of host penetration by the fungal pathogen. Confocal laser scanning microscopy of M. oryzae‐infected susceptible rice cultivars showed the dimorphic growth pattern that is typically observed during the biotrophic and necrotrophic stages of leaf colonization in susceptible cultivars. The suitability of the gfp‐expressing M. oryzae isolate for further research on R‐gene function and identification of resistant genotypes in rice germplasm collections is discussed.     

Germination and appressorium formation of wheat leaf rust on susceptible,partially resistant and resistant wheat seedlings and on seedlings of other Gramineae

Germination and appressorium formation of wheat leaf rust urediospores were studied in two experiments. No consistent differences could be detected between 11 genotypes of wheat (Triticum aestivum), two of barley, one ofTriticum dicoccum, one ofT. dicoccoides, one ofT. boeoticum and one ofAegilops squarrosa. Host genotypes and non-hosts gave similar results, suggesting that the phases before penetration of the host leaf are not affected by the resistance mechanisms operating in hosts and non-host genotypes.Samenvatting Kieming en de vorming van appressoria is bestudeerd in twee experimenten. Er zijn geen systematische verschillen waargenomen tussen 11 genotypen van tarwe, twee van gerst, een vanTriticum dicoccum, een vanT. dicoccoides, een vanT. boeoticum en een vanAegilops squarrosa. Waard en niet-waard genotypen gaven overeenkomstige resultaten, dit suggereert dat de fasen voor penetratie van het gastheerblad niet beïnvloed worden door de resistentiemechanismen werkzaam in waard en niet-waard.     

Agrobacterium-mediated transformation efficiency is altered in a novel rice bacterial blight resistance cultivar and is influenced by environmental temperature

Y73 is a progeny of asymmetric somatic hybridization from an elite japonica rice cultivar (Dalixiang) and a wild rice (Oryza meyeriana), which shows high resistance to bacterial blight. It has a similar genetic background to its recurrent parent, Dalixiang, but Y73 has a high resistance to both bacterial blight and Agrobacterium. The transformation efficiency of Y73 was 35.7%, while Dalixiang had higher transformation efficiency (71.2%) under the same co-cultivation temperature (25 °C). These results indicate that the resistance to Agrobacterium tumefaciens in Y73 was linked with its characteristic of bacterial blight resistance, and was obtained from its donor parent, O. meyeriana. Further studies also showed that the resistance to A. tumefaciens was weakened at a lower temperature (20 °C). To study its molecular mechanism, the expression levels of genes associated with Agrobacterium-mediated transformation (OsMPKs, OsVIP1s and OsPR1s) in rice were investigated by Quantitative real-time PCR (qRT-PCR) analysis. The expression levels of OsMPK genes remained unchanged at different temperatures, while all OsVIP1s and almost all OsPR1s were up-regulated and transformation efficiency of Y73 was increased notably when infected by Agrobacterium at 20 °C compared with 25 °C. This study suggests that the bacterial blight resistance genes in Y73 also have an effect on Agrobacterium-mediated transformation and that the response to temperature is associated with the regulation of MAPK/VIP1 defense signaling pathway.