Flagellin from Pseudomonas syringae pv. tabaci induced hrp-independent HR in tomato

We have previously shown that flagellin of Pseudomonas syringae pv. tabaci is an elicitor that induces a hypersensitive reaction (HR) in nonhost tomato cells. Flagellin is the major HR elicitor produced by this pathogen, as shown by the inability of a flagellin-defective mutant, ΔfliC, to induce HR. Also, a ΔfliD mutant that secretes large amounts of monomer flagellins induces a strong HR in tomato. In this study, the possible involvement of an Hrp type III secretion system (TTSS) in flagellin-induced HR was investigated using flagella-defective mutants or Hrp TTSS-defective mutants. The hrcC gene encodes HrcC protein, which is required for Hrp pilus formation in the outer membrane. An hrcC mutation, introduced into the wild-type, ΔfliC, and ΔfliD mutants of P. syringae pv. tabaci did not affect swimming motility or flagellin secretion, whereas all ΔhrcC, ΔfliC, and ΔfliD mutants lost the ability to cause disease on host tobacco leaves. However, the ΔhrcC mutant and the ΔfliD/ΔhrcC double mutant were still able to induce HR cell death, expression of one of the defense-related genes hsr203J, and the generation of hydrogen peroxide in nonhost tomato cells. Thus, flagellin is required for both pathogenicity in host tobacco and HR in nonhost tomato. On the other hand, hrp TTSS is necessary for pathogenicity on host tobacco but is not indispensable to induce HR in nonhost tomato. These results clearly show that flagellin-induced HR is hrp-independent in tomato.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB049570     

Transgenic indica rice expressing a bitter melon (Momordica charantia) class I chitinase gene (McCHIT1) confers enhanced resistance to Magnaporthe grisea and Rhizoctonia solani

McCHIT1 chitinase (DQ407723), a class I secretory endochitinase from bitter melon (Momordica charantia), had been demonstrated to enhance resistance against Phytophthora nicotianae and Verticillium wilt in transgenic tobacco and cotton. In order to obtain disease-resistant transgenic rice, McCHIT1 was transformed into a restorer line JinHui35 (Oryza sativa subsp. indica) by using the herbicide-resistance gene Bar as the selection marker. Transgenic rice lines and their progenies overexpressing the McCHIT1 gene showed enhanced resistance to Magnaporthe grisea (rice blast) and Rhizoctonia solani (sheath blight), two major fungal pathogens of rice. McCHIT1-transgenic rice confirmed the inheritance of the transgene and disease resistance to the subsequent generation. The T₂ transformants exhibited significantly increased tolerance to M. grisea, with a 30.0 to 85.7 reduction in disease index, and R. solani, with a 25.0 to 43.0 reduction in disease index, based on that of the control as 100. These results indicated that over-expression of the McCHIT1 gene could lead to partial disease reduction against these two important pathogens in transgenic rice.     

黑龙江省部分地区稻瘟病菌致病性分析及鉴别体系优化

为明确哈尔滨市及鸡西市稻瘟病菌致病性变化情况并优化鉴别体系,以24个水稻单基因系为鉴别体系,对2006—2008年采自2市的稻瘟病菌菌株进行致病型划分,并应用聚类分析方法完成鉴别体系优化。结果表明,应用水稻单基因系鉴别体系可将哈尔滨市2006—2008年采集的稻瘟病菌菌株划分为34、12和27个致病型,致病性相似系数分别为0.32~1.00、0~1.00和0.20~1.00;水稻单基因系对其抗性频率分别为2.86%~97.14%、8.33%~100.00%和10.00%~96.67%;鸡西市2006—2008年采集的菌株可划分为26、19和20个致病型,致病性相似系数分别为0.47~0.92、0.15~0.86和0.39~1.00;水稻单基因系对其抗性频率分别为3.85%~96.15%、5.26%~73.68%和4.76%~95.24%。适用于哈尔滨市的优化鉴别体系包括Pi-9、Pi-11、Pi-a、Pi-ks、Pi-5、Pi-i、Pi-sh、Pi-3、Pi-km和Pi-ta共10个基因,2006—2008年累计方差贡献率分别为86.15%、98.77%和87.39%;适用于鸡西市的优化鉴别体系包括Pi-9、Pi-11、Pi-a、Pi-ks、Pi-5、Pi-i、Pi-sh、Pi-1、Pi-7和Pi-t共10个基因,2006—2008年累计方差贡献率分别为82.58%、94.55%和90.37%。应用优化后的鉴别体系可将哈尔滨市2006—2008年采集的菌株划分为22、12和23个致病型,将鸡西市2006—2008年采集的菌株划分为22、14和18个致病型。     

Histological characterization of the early-stage infection events of Setosphaeria turcica in maize

Northern corn leaf blight (NCLB) caused by Setosphaeria turcica is a major foliar disease of maize. The early-stage infection events of this pathogen on maize leaves are unclear. We investigated the optimum temperature for conidial germination and appressorium formation, and characterized penetration and growth of S. turcica in maize leaf sheath and onion epidermis cells, including use of histological staining to assess plant cell viability. The results showed that the optimum temperature for conidial germination and appressorium formation was 20°C. On the maize leaf sheath, the appressoria were formed by germinated conidia, and penetration on the epidermal cells occurred at 8 h postinoculation (hpi). Round vesicles developed beneath the appressoria. Between 16 and 24 hpi, the branched invasive hyphae invaded three to five adjacent cells at most infection sites. The invasive hyphae tended to move along the cell wall and crossed from one cell to another. In the onion epidermis cells, the appressoria formed at 8 hpi, and in most cases the epidermal cells were penetrated through the juncture of the cell walls. At 16–24 hpi, the primary hyphal terminus swelled to a vesicle. The maize leaf sheath cells died at 8 hpi, whereas the onion cells did not. Our findings documented in detail the penetration and invasive hyphal growth in maize leaf sheath and onion epidermis, as well as viability of plant cells, at the early stages of infection, and provide a foundation for elucidating the underlying mechanism of S. turcica–maize interactions.     

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.     

水稻纹枯病生防细菌筛选及其与病原菌侵染垫形成的关系

为筛选对立枯丝核菌Rhizoctonia solani具有拮抗作用的生防细菌,利用稀释涂布平板法,从吉林省水稻纹枯病样中分离筛选出对立枯丝核菌AG-1A具有高拮抗活性的生防菌株,通过gyrB基因序列分析鉴定其分类地位,并采用离体侵染试验和温室防治试验测定筛选生防菌株对水稻纹枯病的防效,在显微镜下观察生防菌株预处理后接种病原菌的水稻叶片表面的菌丝生长情况,分析其生防机理。结果显示,从水稻纹枯病样中共分离获得35株菌株,其中菌株ND11对立枯丝核菌AG-1A的抑制率和抑菌圈直径最大,分别为74.12%和31.50 mm。基于gyrB基因序列分析最终将菌株 ND11鉴定为短小芽胞杆菌Bacillus pumilus;该菌株能够减缓立枯丝核菌AG-1A的侵染速度,抑制其侵染垫的形成,对水稻纹枯病的温室防效达70.69%以上。表明短小芽胞杆菌菌株ND11能够通过抑制立枯丝核菌侵染垫的形成来防治水稻纹枯病,且防效较好,具有开发为生防菌剂的潜力。     

Phyllosphere yeasts antagonize penetration from appressoria and subsequent infection of maize leaves by Colletotrichum graminicola

In growth cabinet experiments, the common phyllosphere yeastsSporobolomyces roseus andCryptococcus laurentii var.flavescens were sprayed as a mixture (11) onto the fourth leaves of maize plants (Zea mays) two-three days prior to inoculation withColletotrichum graminicola. In four experiments the average yeast population of the treated leaves at the time of pathogen inoculation varied between 5× 10⁴ and 8× 10⁵ cells cm^–2 leaf, whereas on the untreated leaves the yeast population varied from <10³ to 10⁴ cells cm^–2 leaf. The yeasts reduced lesion density and necrosis fromC. graminicola infection by approximately 50%. Contrary to findings with other necrotrophic pathogens, conidial germination, superficial mycelial growth and appressorium formation were not affected. Instead, the reduction of infection could only be explained by a reduced number of penetrations from the normally formed appressoria, a site of interaction not previously recorded.Samenvatting In klimaatkastexperimenten werden maisbladeren (4e blad) twee-drie dagen voor inoculatie metColletotrichum graminicola bespoten met een mengsel (11) van de algemeen voorkomende fyllosfeergistenSporobolomyces roseus enCryptococcus laurentii var.flavescens. In vier experimenten varieerde de gemiddelde gistpopulatie op de behandelde bladeren, op het moment van inoculatie met het pathogen, van 5× 10⁴ tot 8× 10⁵ cellen cm^–2 blad, op de onbehandelde bladeren van <10³ tot 10⁴ cellen cm^–2 blad. De gisten reduceerden de lesiedichtheid en het necrotisch bladoppervlak tengevolge van deC. graminicola infectie voor ongeveer 50%. De stadia in de ontwikkeling van andere necrotrofe pathogenen, die gewoonlijk gevoelig zijn voor antagonisme door gisten, zoals sporekieming, oppervlakkige myceliumgroei en vorming van appressoria, werden bijC. graminicola niet beïnvloed. De waargenomen reductie van infectie kon alleen verklaard worden door een remming van de penetratie vanuit normaal gevormde appressoria. Interactie in dit stadium van het infectieproces is nog niet eerder waargenomen.     

Defense responses of Arabidopsis thaliana inoculated with Pseudomonas syringae pv. tabaci wild type and defective mutants for flagellin (ΔfliC) and flagellin-glycosylation (Δorf1)

Flagellin, an essential component of the bacterial flagellar filament, is capable of inducing a hypersensitive response (HR), including cell death, in a nonhost plant. A flagellin-defective mutant (ΔfliC) of Pseudomonas syringae pv. tabaci lacks both the flagellar filament and motility, whereas a flagellin-glycosylation-defective mutant (Δorf1) retains the flagellar filament but lacks the glycosyl modification of flagellin protein. To investigate the role of flagellin protein and its glycosylation in the interaction with its nonhost Arabidopsis thaliana, we analyzed plant responses after inoculation with these bacteria. Inoculation with wild-type P. syringae pv. tabaci induced HR, with the generation of reactive oxygen species and cell death. In contrast, inoculation with either ΔfliC or Δorf1 mutant induced a low level of HR, and inoculated leaves developed a disease-like yellowing. These mutant bacteria multiplied better than the wild-type bacteria in A. thaliana. These results indicate that A. thaliana expresses a defense reaction in response to the bacterial flagellin with its glycosyl structure.     

Appresoorium formation of Puccinia hordei on partially resistant barley and two non-host species

One of the components of partial resistance of barley to leaf rust,Puccinia hordei, is a reduced infectibility. It was investigated whether this low infectibility may rest on a hampered appressorium formation of the leaf rust fungus. The appressorium formation on the primary leaves of 11 barley genotypes with an intermediate-to-low infectibility was compared with that on the highly infectible L94. The number of stomata per cm² leaf area occupied by appressoria ofP. hordei was determined per genotype by means of fluorescence microscopy. No cosistent differences could be detected, indicating that the mechanisms causing a low infectibility of partially resistant barley seedlings act at a phase later than the formation of the appressoria. On the non-host wheat not fewer appressoria were formed than on L94, but no appressoria were found on a lettuce genotype. The latter probably lacks the stimuli that enable the fungus to find stomata.Samenvatting Eén van de componenten van partiële resistentie van gerst tegen dwergroest,Puccinia hordei, is een verminderde infectiedichtheid. Het mechanisme, dat hieraan ten grondslag ligt, is onbekend. Een experiment werd uitgevoerd om na te gaan of bij partieel resistente rassen een verminderde appressoriumvorming optreedt. Na inoculatie in een inoculatietoren en een zorgvuldig uitgevoerde incubatie werd het aantal huidmondjes per cm² bladoppervlak bepaald dat bezet was door appressoria vanP. hordei. De elf weinig vatbare gerstlijnen uit deze studie bleken niet reproduceerbaar te verschillen van de zeer vatbare gerstlijn L94 in de mate van appressoriumbezetting. Dit wijst erop dat infectiedichtheidsverschillen t.g.v. partiële resistentie veroorzaakt worden door mechanismen die werken na de appressoriumvorming. In een tweede experiment werd aangetoond dat zelfs op de niet-waardsoort tarwe, waaropP. hordei geen symptomen veroorzaakt, niet minder appressoria worden gevormd dan op L94. Op een sla-genotype trad echter geen appressoriumvorming op. Op deze laatste niet-waardsoort ontbreken waarschijnlijk de stimuli die de schimmel in staat stellen huidmondjes te vinden.     

The DNA damage signal transducer ortholog <Emphasis Type="Italic">Mop53BP1</Emphasis> is required for proper appressorium differentiation and pathogenicity in <Emphasis Type="Italic">Pyricularia oryzae</Emphasis>

Appressorium differentiation, one of the most important steps in pathogenesis by the rice blast fungus, Pyricularia oryzae, is strongly coordinated with the cell cycle. In this study, we identified an ortholog gene of 53BP1, which encodes a signal transducer protein that participates in G2-M cell cycle checkpoint in higher eukaryotes, in the genome of P. oryzae and characterized the phenotype of deletion and overexpression mutants. Deletion mutants showed no significant deficiency in vegetative growth compared to wild-type and complemented strains, even on the media containing DNA-damaging agents. However, these null mutants had abnormal appressoria and formed more appressoria per conidium than in the wild type and were unable to penetrate the epidermis of rice leaves. eGFP-fused Mop53BP1 and qRT-PCR analyses revealed that Mop53BP1 is expressed during the first hours of appressorium formation. In addition, in overexpression mutants, Mop53BP1 localized to nuclei during all stages of appressorium maturation and penetration, and the mutants were resistant to the microtubule inhibitor benomyl, suggesting that Mop53BP1 is nuclear protein and may have some role related to microtubules.     

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.     

Recovery and characterization of asexual recombinants ofMagnaporthe grisea

Eleven nitrate non-utilizing (nit) mutants were recovered from six field isolates ofMagnaporthe grisea that had different degrees of sensitivity to the blasticide kitazin P (iprobenfos, IBP). Allnit mutants were resistant to chlorate and there were no significant differences in hyphal growth rate, conidial production, sensitivity to IBP, and pathogenicity betweennit mutants and their parent isolates.nit phenotypes and IBP-resistance were used as two independent genetic markers to study asexual recombination inM. grisea. Asexual recombinants were recovered from the heterokaryotic mycelium of three inter-strain pairings, namely, DY2-3(nitl-LR) + A7-3 (nitM-S); A7-3 (nitM-S) + F4-2 (nitl-MR); and F4-2 (nitl-MR) + DY2-4 (nitA-LR), at a frequency of 7.31%, 14.00% and 8.63%, respectively. The growth rate, conidial production and pathogenicity of asexual recombinants were similar to those of parental strains. We concluded that asexual recombination resulting from hyphal fusion might contribute to variability inM. grisea. http://www.phytoparasitica.org posting Nov. 14, 2005.     

Synergistic induction of wheat stem rust appressoria by chemical and topographical signals

The leaf alcohols cis -3-hexen-1-ol and trans -2-hexen-1-ol have been previously shown to induce the wheat stem rust (Puccinia graminis f. sp. tritici) to differentiate appressoria in a complex axenic culture medium. In this paper, the possible role of these chemicals in appressorium induction during infection has been analysed further. The inductive potential of ethylene and three cutin monomers were also tested because they have been found to induce appressoria in other fungi. Both hexenols were found to be inductive in aqueous solution in the absence of media constituents. 0.5–1 m trans -2-hexen-1-ol was the most inductive resulting in a maximum of 51% appressorium differentiation. trans -2-hexen-1-ol was also shown to be inductive in vapour form. In aqueous solution, trans -2-hexen-1-ol acted synergistically with topographical signals by increasing the rate of appressorium induction. Combining the two signals also increased the total number of appressoria differentiated (88%). The other chemicals tested were non-inductive although a biochemical domain rich in exposed cutin was localized around stomatal apertures of wheat leaves. The characteristic bipolar morphology of appressoria formed over stomata was found to be determined by topographical signals. Overall, our data support a role for both chemical signals (hexenols or their analogues) and topographical signals being involved in appressorium induction by wheat stem rust.     

福建省稻瘟病菌对主要抗瘟基因及主栽品种的致病性分析

为明确福建省各稻区的主要抗瘟基因及主栽品种的利用价值,2012—2014年采用喷雾法测定了丽江新团黑谷上的193个菌株对30个水稻抗瘟基因及93个主栽水稻品种的致病性。结果表明,供试稻瘟病菌对30个抗瘟基因表现为强致病力和较强致病力的出现频率分别为13.47%和52.85%,对93个主栽品种表现为强致病力和较强致病力的出现频率分别为1.55%和11.40%;供试稻瘟病菌对抗瘟基因Pi-k~m、Pi-7(t)、Pi-k~p和Pi-9(t)的毒力频率均低于20%;供试稻瘟病菌对谷优2329、谷优5138、昌优964等37个品种的毒力频率均低于20%,对谷优系列、全优系列、深优系列、泰丰优系列及天优系列水稻品种的毒力频率均低于20%。研究表明,在福建地区抗瘟基因Pik~m、Pi-7(t)、Pi-k~p和Pi-9(t)可作为抗源使用,且谷丰A、全丰A、深97A、泰丰A和天丰A仍是抗瘟性较好的育种亲本材料。     

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。     

Control of rice sheath blight by phyllosphere epiphytic microbial antagonists

Epiphytic microorganisms on the phyllosphere of traditional and high-yielding rice varieties were isolated from different agroecological zones of Sri Lanka and screened for theirin vitro andin vivo antagonism againstRhizoctonia solani Kühn AG-1 1A, the sheath blight pathogen of rice. Among a total of 196 bacterial and 91 fungal isolates, 12 bacterial and two fungal isolates which showed more than 50% growth inhibition ofR. solani were tested for theirin vivo antagonism. Among the 14 antagonists tested, six bacterial and one fungal isolate substantially reduced the incidence of sheath blight (by more than 82%) and severity (by more than 92%) of the rice varieties BG94-1 and IR8 grown in a pot experiment under open field conditions. Using five antagonists that showed the bestin vitro antagonism, a pot experiment was conducted to determine whether the presence of indigenous microflora on the rice sheath had any effect on the effectiveness of antagonism. Three isolates (B4, GbB5 and HMWB4) controlled sheath blight incidence and severity equally well in the presence and absence of indigenous microflora. Two isolates (BG352B1 and BG300B1) were more effective when they were introduced into the rice sheath without indigenous microflora. Among the effective antagonists determined by the pot experiment, isolates B4, B16, BG94-1B5, GbB5, HMWB4 and BG379-F2 were tested under field conditions for two consecutive growing seasons. Under field conditions, severity of rice sheath blight was significantly reduced by the application of all the tested antagonists as a spray on rice sheath at a concentration of 10⁸ cfu ml⁻¹, starting 3 days after the development of symptoms and continuing for three applications at 10-day intervals. Antagonistic performances were consistent in the two seasons under field conditions andB. megatarium A (isolate B16) andAspergillus niger (isolate BG379-F2) performed as the most effective antagonists in both seasons. When disease severity was quantified as percentage sheath area covered by the disease lesions, the respective reductions in disease severity were greater than 50% and 61% byB. megaterium A (isolate B16) andAspergillus niger (isolate BG379-F2), respectively, in both seasons. http://www.phytoparasitica.org posting Jan. 10, 2008.     

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.     

Pi34-AVRPi34: a new gene-for-gene interaction for partial resistance in rice to blast caused by Magnaporthe grisea

The japonica rice (Oryza sativa) cultivar Chubu 32 has a high level of partial resistance to blast, which is mainly controlled by a dominant resistance gene located on chromosome 11. The partial resistance to the rice blast fungus (Magnaporthe grisea) in Chubu 32 has isolate specificity; isolate IBOS8-1-1 is more aggressive on Chubu 32 than are other isolates. We hypothesized that the gene-for-gene relationship fits this case of a partial resistance gene in Chubu 32 against the avirulence gene in the pathogen. The partial resistance gene in Chubu 32 was mapped between DNA markers C1172 (and three other co-segregated markers) and E2021 and was designated Pi34. In the 32 F₃ lines from the cross between a chromosome segment substitution line (Pi34⁻) from Koshihikari/Kasalath and Chubu 32, the lines with high levels of partial resistance to the M. grisea isolate Y93-245c-2 corresponded to the presence of Pi34 estimated by graphic genotyping. This indicated that Pi34 has partial resistance to isolate Y93-245c-2 in compatible interactions. The 69 blast isolates from the F₁ progeny produced by the cross between Y93-245c-2 and IBOS8-1-1 were tested for aggressiveness on Chubu 32 and rice cultivar Koshihikari (Pi34⁻). The progeny segregated at a 1 : 1 ratio for strong to weak aggressiveness on Chubu 32. The results suggested that Y93-245c-2 has one gene encoding avirulence to Pi34 (AVRPi34), and IBOS8-1-1 is extremely aggressive on Chubu 32 because of the absence of AVRPi34. This is the first report of a gene-for-gene relationship between a fungal disease resistance gene associated with severity of disease and pathogen aggressiveness.     

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.     

DNA fragmentation in Sekiguchi lesion mutants of rice infected with Magnaporthe grisea

Light-dependent activation of the tryptamine pathway in Sekiguchi lesion (sl) mutants (Sekiguchi-asahi, Sekiguchi-koshihikari, and Sekiguchi-sasanishiki) inoculated with Magnaporthe grisea, was demonstrated by a significant increase in tryptophan decarboxylase and monoamine oxidase activities and tryptamine accumulation. Terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) staining and gel analysis indicated DNA fragmentation in cells of leaf tissues with Sekiguchi lesions of the three sl mutants. Furthermore, increased DNase activity was also light-dependent in the sl mutants after inoculation with M. grisea. DNA fragmentation was inhibited in leaves when Sekiguchi lesion formation was suppressed by cycloheximide and heat shock pretreatments. These data suggest that Sekiguchi lesion formation in the sl mutants is an apoptosis-like response and that its response is induced by light-dependent activation of the tryptamine pathway, which is responsible for light-enhanced resistance.The nucleotide sequence data reported are available in the DDBJ/GenBank/EMBL databases under accession number AB162137 for TDC