Pea extracellular Cu/Zn-superoxide dismutase responsive to signal molecules from a fungal pathogen

We previously reported that the release of O₂ ⁻ from isolated pea cell walls was enhanced by a 70-kDa glycoprotein elicitor but was suppressed by mucin-type glycopeptide suppressors (supprescins A and B) prepared from pycnospore germination fluid of Mycosphaerella pinodes, causal agent of Mycosphaerella blight of pea. Here, we show that superoxide dismutase (SOD) in the apoplast fluid/cell wall of pea seedlings responds to the fungal elicitor and suppressor molecules. In a pharmacological study and with internal amino acid sequencing, the apoplastic SOD in a pea cultivar Midoriusui was found to be a Cu/Zn type SOD. We cloned a full-length cDNA of the Cu/Zn-SOD and designated it as PsCu/Zn-SOD1. An increase in PsCu/Zn-SOD1 mRNA and the PsCu/Zn-SOD1 protein was induced by treatment with the elicitor more intensively than by wounding. Such induction by the elicitor or wounding, however, was inhibited by the concomitant presence of supprescins. The SOD activity of recombinant PsCu/Zn-SOD1 was regulated directly by these signal molecules in a manner similar to their effect on the SOD activity in the apoplastic fluid and in the cell wall-bound proteins. Based on these findings, we discuss a role for PsCu/Zn-SOD1 in the pea defense response. The nucleotide sequence data of PsCu/Zn-SOD1 reported are available in the DDBJ/EMBL/GenBank databases under accession number AB189165.     

A binding protein for fungal signal molecules in the cell wall of Pisum sativum

In the plant cell wall of Pisum sativum seedlings, we found an NTPase (E.C. 3.6.1.5.) with ATP-hydrolyzing activity that was regulated by an elicitor and suppressors of defense from pea pathogen Mycosphaerella pinodes. The ATPase-rich fraction was purified from pea cell walls by NaCl solubilization, ammonium sulfate precipitation, and chromatography with an ATP-conjugated agarose column and an anion-exchange column. The specific activity of the final ATPase-rich fraction increased 600-fold over that of the initial NaCl-solubilized fraction. The purified ATPase-rich fraction also had peroxidase activity and generated superoxide, both of which were regulated by the M. pinodes elicitor and suppressor (supprescins). Active staining and Western blot analysis also showed that the ATPase was copurified along with peroxidases. In this fraction, a biotinylated elicitor and the supprescins were bound primarily and specifically to ca. 55-kDa protein (CWP-55) with an N-terminal amino acid sequence of QEEISSYAVVFDA. The cDNA clone of CWP-55 contained five ACR domains, which are conserved in the apyrases (NTPases), and the protein is identical to a pea NTPase cDNA (GenBank accession AB071369). Based on these results, we discuss a role for the plant cell wall in recognizing exogenous signal molecules.     

Defense strategies of pea embryo axes with different levels of sucrose to Fusarium oxysporum and Ascochyta pisi

The aim of this study was to compare the defense responses of embryo axes of Pisum sativum L. cv. Kwestor with different sucrose levels to pathogenic fungi, i.e. systemic acting Fusarium oxysporum f. sp. pisi and locally acting Ascochyta pisi. Embryo axes were cultured on Heller medium for 96 h. Four variants were compared: these included inoculated embryo axes cultured with or without 60 mM sucrose (+Si and −Si) and non-inoculated embryo axes cultured with or without 60 mM sucrose (+Sn and −Sn). After inoculation of the pea embryo axes with pathogenic fungi a generally higher concentration of free radicals was detected by electron paramagnetic resonance (EPR), in comparison to non-inoculated embryo axes. The inoculation with F. oxysporum caused stronger generation of free radicals in −Si than in +Si embryo axes. A different response was observed after inoculation with A. pisi; starting from 48 h, the concentration of free radicals in +Si axes was found to be 1.5 times higher than in −Si embryo axes. The values of spectroscopic splitting coefficients for these radicals suggest that they are semiquinone radicals. The EPR method also revealed Mn²⁺ ion accumulation after 24 h of culture. Over time, high levels of these ions were recorded in +Si embryo axes inoculated with F. oxysporum, while in +Si embryo axes inoculated with A. pisi they decreased. Up to 48 h after inoculation with the pathogenic fungi, Mn²⁺ ion levels were higher in +Si embryo axes than in +Sn axes. The activity of superoxide dismutase (SOD, EC 1.15.1.1) increased in +Si embryo axes up to 72 h after inoculation with pathogenic fungi; however, it was generally lower than in +Sn axes. Catalase activity (CAT, EC 1.11.1.6) increased up to 72 h after inoculation with F. oxysporum and the values were higher than in the non-inoculated tissue. Especially high activity of this enzyme was noted in −Si embryo axes after inoculation with either F. oxysporum or A. pisi. Peroxidase activity (POX, EC 1.11.1.7) towards pyrogallol in embryo axes increased during culture; however, it was lower or similar to that in non-inoculated embryo axes. SOD, CAT and POX zymograms showed that the synthesis of new isoforms was induced after inoculation with pathogenic fungi. Peroxidase isozymes detected by the reaction with diaminobenzidine in native PAGE were intensely stained in +Si embryo axes after inoculation with pathogenic fungi. Respiratory activity of the inoculated tissues was considerably higher than in non-inoculated tissues. The respiration rate was generally much higher in +Si than in −Si embryo axes. Growth of −Si embryo axes was more significantly retarded as a consequence of inoculation than that of +Si embryo axes.These results indicate that, depending on the manner of influence of a pathogenic fungus, both similar and differing defensive strategies may be initiated and a raised sugar levels in pea tissues limit the development of F. oxysporum and A. pisi.     

Localization and responsiveness of a cowpea apyrase VsNTPase1 to phytopathogenic microorganisms

Apyrases (NTPases) are associated with both compatible and incompatible interactions between plants and microorganisms. Previously we reported that the ATPase activities of cell-wall-bound apyrases of several leguminous plants, such as pea, cowpea, soybean, and kidney bean, were enhanced by a glycoprotein elicitor and were inhibited in a species-specific manner by mucin-type glycopeptide suppressors secreted from a pea pathogenic fungus, Mycosphaerella pinodes. In this study, we isolated two apyrase genes, VsNTPase1 and VsNTPase2, from a cDNA library of Vigna sinensis Endl. cv. Sanjakusasage. Based on phylogenetic analysis, VsNTPase1 may belong to a group that responds to environmental stimuli. In a transient assay using DNA bombardment, a fusion protein of green fluorescent protein (GFP) and the N-terminal putative signal sequence of VsNTPase1 was distributed in the nucleus, cytoplasm (cytoskeletal structure), and cell wall. On the other hand, a fusion protein of GFP and the N-terminal putative VsNTPase2-signal sequence was localized in the cytoplasm, especially in small particles (perhaps mitochondria). A recombinant VsNTPase1 expressed in Spodoptera frugiperda 21 cells responded directly to signal molecules from several phytopathogenic microorganisms. Here, we discuss the role of apyrases in recognizing and responding to exogenous signals. The nucleotide sequences of VsNTPase1 and VsNTPase2 in this article have been submitted to DDBJ as accession numbers AB196769 and AB196770, respectively.     

Dry mycelium of<Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> induces resistance against verticillium wilt and enhances growth of cotton plants

Dry mycelium (DM) ofPenicillium chrysogenum and its water extract (DME) were examined for their effects on induced resistance against Verticillium wilt and plant growth of cotton in the greenhouse. Soil application of 0.1–5% DM or 0.5–5% DME provided significant protection against the wilt, relative to the control. As neither DM nor DME inhibited mycelial growth ofVerticillium dahliae in vitro, it is suggested that the disease-controlling effects of DM or DME are attributed to induced resistance. DME (5%), as well as DME treated with chloroform or cold acetone, were as effective as 2% DM in reducing disease severity of Verticillium wilt, implying that the resistance-inducing substance(s) in DM are mostly water-soluble, with neither proteins nor lipids likely to be responsible for the induction of resistance. No significant difference in root colonization withV. dahliae was found between control-inoculated and 2% DM- or 5% DME-inoculated plants. However, colonization of hypocotyls and epicotyls was drastically suppressed by either 2% DM or 5% DME relative to the control. Treatments with 2% DM or 5% DME significantly increased ionically-bound peroxidase (POX) activity in roots, hypocotyls and the second leaf of cotton plants, with the hypocotyls expressing the highest increase. Soil application of DM or DME increased plant height, fresh and dry weight of inoculated and non-inoculated cotton plants, relative to their corresponding controls. It is concluded that DM may be used in cotton crops to promote plant growth and to induce resistance againstV. dahliae. POX might be associated with the defense against Verticillium wilt. http://www.phytoparasitica.org posting Jan. 9, 2002.     

<Emphasis Type="Italic">Eremothecium coryli</Emphasis> and <Emphasis Type="Italic">E.</Emphasis><Emphasis Type="Italic">ashbyi</Emphasis> cause yeast spot of azuki bean

Yeast-like fungi were isolated from lesions on azuki bean (cv. Shin-Kyotodainagon) seeds that had been sucked by bean bugs in Kyoto Prefecture, Japan. On the basis of morphological and physiological characteristics and sequence data of the internal transcribed spacer (ITS) regions including the 5.8S rDNA, these yeasts were identified as Eremothecium coryli and E. ashbyi. Pathogenicity of those yeasts was confirmed by a reinoculation test. To our knowledge, this is the first report of the occurrence of yeast spot in azuki bean in Japan. The nucleotide sequence data reported are available in the GeneBank/EMBL/DDBJ database as accessions AB478291–AB478309 for E. coryli AZC1–19 and AB478310–AB478317 for E. ashbyi AZA1–8.     

蚕豆和豌豆锈病病原菌的分子鉴定

为明确我国热带和亚热带地区蚕豆Vicia faba和豌豆Pisum sativum锈病的病原菌种类,通过致病性测定和ITS序列系统发育分析对来自我国云南省玉溪市的4份豌豆锈菌分离物及云南、广西、重庆和四川省(区、市)的5份蚕豆锈菌分离物进行系统鉴定。结果显示,分离自豌豆的锈菌WX1分离物对蚕豆和豌豆均具有高致病性,在侵染叶片上产生大量锈子器;分离自蚕豆的锈菌CX3分离物仅对蚕豆具有高致病性,能在叶片上产生大量夏孢子,而对豌豆的致病性相对较低,仅产生少量的夏孢子堆;分离物WX1和CX3对小扁豆和鹰嘴豆不具有致病性。基于ITS序列系统发育分析表明,所有不同寄主来源的蚕豆单胞锈菌分离物均聚类于一个系统发育组,但分离自蚕豆和豌豆的分离物分别聚类在不同的亚组。表明分离自云南省玉溪市豌豆上的蚕豆单胞锈菌Uromyces viciae-fabae应为豌豆专化型,定名为U. viciae-fabae ex P. sativaum,而来源于云南、广西、重庆和四川省(区、市)的蚕豆锈病病原菌为蚕豆专化型U. viciae-fabae ex V. faba。     

Bacterial induction of the accumulation of phaseollin,pisatin and rishitin and their antibacterial activity

Bean hypocotyls, pea pods and tomato fruits were tested for phaseollin, pisatin and rishitin production when challenged with the phytopathogenic bacteriaErwinia carotovora, Pseudomonas phaseolicola, P. pisi andP. solanacearum, and their isolated extracellular polysaccharides. All bacteria induced phytoalexin accumulation, whereas only phaseollin and pisatin, but not rishitin, were elicited by EPS. The inhibitory effect of these three phytoalexins on bacterial growth was studied in liquid medium; whereas phaseollin and pisatin strongly inhibited growth, only a slight inhibitory effect resulted from the presence of rishitin in the medium.Samenvatting Bonehypocotylen, erwtepeulen en tomatevruchten werden onderzocht op hun vermogen tot vorming van respectievelijk faseolline, pisatine en rishitine, na inoculatie met de fytopathogene bacteriënErwinia carotovora, Pseudomonas phaseolicola, P. pisi enP. solanacearum en na behandeling met oplossingen van hun extracellulaire polysacchariden (EPS). Alle bacteriesoorten induceerden fytoalexinevorming, terwijl hun EPS wel faseolline- en pisatine-, maar geen rishitinevorming induceerden. Faseolline en pisatine remden de groei van de bacteriën in vloeibaar medium sterk; rishitine daarentegen had slechts een geringe groeiremming tengevolge.     

Reduced consumption of pea protein-treated flour disks by stored-product insects

Consumption of pea (Pisum sativum L.) protein-treated wheat flour by the red flour beetleTribolium castaneum (Herbst.), the rice weevilSitophilus oryzae (L.) and the lesser grain borerRhyzopertha dominica (F.), was significantly reduced compared with wheat flour alone. Consumption was affected when the insects were exposed for 3 days to flour disks containing protein-rich fraction of the ‘Bonneville’ pea variety. Antifeedants present in the pea protein fraction are apparently responsible for the reduced feeding response in these species. http://www.phytoparasitica.org posting May 6, 2004.     

Plant Host Range of Verticillium longisporum and Microsclerotia Density in Swedish Soils

Verticillium longisporum is a soil-borne fungal pathogen causing vascular wilt of Brassica crops. This study was conducted to enhance our knowledge on the host range of V. longisporum. Seven crop species (barley, oat, oilseed rape, pea, red clover, sugar beet and wheat) and five weed species (barren brome, black-grass, charlock, cleavers and scentless mayweed) all common in southern Sweden were evaluated for infection by response to V. longisporum. Oat, spring wheat, oilseed rape, scentless mayweed and charlock inoculated with V. longisporum in a greenhouse showed stunting to various degrees close to the fully ripe stage. Based on the extent of microsclerotia formation, explants were separated into four groups: for pea and wheat, <5% of the samples had formed microsclerotia; for scentless mayweed, 5–10%; for oat, 10–20%; and for charlock and oilseed rape >80%. The results suggest that plant species outside the Brassicaceae can act as reservoirs of V. longisporum inoculum. Soil inoculum densities in nine fields were monitored over a period of 12 months, which ranged from 1 to 48 cfu g⁻¹ soil. Density of microsclerotia was lowest just after harvest, reaching its maximum six months later. No significant correlation between inoculum density in soil and disease incidence on oilseed rape plants was found. However, the data suggest that a threshold of 1 cfu g⁻¹ soil is needed to cause disease on oilseed rape. Species identification based on microsclerotia morphology and PCR analysis showed that V. longisporum dominated in soil of seven, and V. dahliae in two of the nine fields studied.     

Identification and pathogenicity of Pythium species causing damping-off of kidney bean

Five Pythium species (Pythium irregulare, P. mamillatum, P. myriotylum, P. spinosum and P. ultimum var. ultimum) were isolated from the hypocotyls and roots of kidney bean plants with damping-off from a commercial field and from experimental plots that have undergone either continuous cropping with kidney bean or rotational cropping with arable crops. In inoculation tests, all five Pythium species were pathogenic to kidney bean. This is the first report of damping-off of kidney bean caused by Pythium species; we named this disease damping-off of kidney bean. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB291811, AB291944 and AB291945.     

Chitinase andβ1,3-glucanase isoforms expressed in pea roots inoculated with arbuscular mycorrhizal or pathogenic fungi

Chitinases were studied in an endomycorrhiza-resistant mutant and wild type pea (Pisum sativum L. cv. Frisson) in order to characterize plant hydrolases specific to pathogenic (Aphanomyces euteiches andChalara elegans) or mycorrhizal (Glomus mosseae) root interactions. Stimulation of constitutive and induction of new chitinase activities was detected by native PAGE for acidic proteins in both pea genotypes inoculated with pathogenic fungi. In contrast, a different additional chitinase isoform was induced inG. mosseae-colonized roots. This isoform was also not elicited in chemically-stressed roots, confirming its mycorrhiza-specificity. Investigations of basic chitinase and-1,3-glucanase activities provided further evidence for differential pea responses during pathogenic and symbiotic interactions.     

Regulation of elicitin-induced ethylene production in suspension-cultured tobacco BY-2 cells

INF1 elicitin, a proteinaceous elicitor produced by Phytophthora infestans, induces a hypersensitive response in tobacco BY-2 cells. In response to elicitin, tobacco cells produce both reactive oxygen species (ROS) and ethylene (ET). To investigate the regulation of elicitin-induced ET production, we pharmacologically analyzed the effects of several chemicals on ET production. Inhibitors of ROS generation or ROS chelators efficiently inhibited ET production, whereas simultaneous treatment of a superoxide anion-generating system with salicylhydroxamic acid recovered ET production. In an in vitro experiment, superoxide anion was necessary and sufficient for conversion of 1-aminocyclopropane-1-carboxylate (ACC) to ET because ET was produced from ACC solely in the presence of the superoxide-generating chemical KO₂. ET production was also inhibited by lipoxygenase (LOX) inhibitors, indicating a possible involvement of LOX-mediated generation of superoxide anion and ET production itself. Furthermore, elicitin-induced ET production was completely inhibited by the protein synthesis inhibitor cycloheximide but recovered after exogenous application of ACC, indicating that de novo protein synthesis is required for ACC accumulation, leading to ET production. We also investigated the effects of several phytohormones on elicitor-induced ET production and discuss their role in the defense response.     

五种杀虫剂对苜蓿种子田中主要害虫和传粉昆虫的影响

为筛选高效、低毒的杀虫剂，测定1.5%除虫菊素、0.3%印楝素、4%阿维·啶虫脒、2.5%高效氯氟氰菊酯和70%吡虫啉5种杀虫剂对苜蓿种子田主要害虫牛角花齿蓟马Odontothrips loti和豌豆蚜Acyrthosiphon pisum的室内毒力和田间防效，并分析这5种药剂对主要传粉昆虫数量及多样性指数的影响。结果显示，0.3%印楝素对牛角花齿蓟马和豌豆蚜的毒力相对较高，LC₅₀分别为1.093 mg/L和2.864 mg/L。70%吡虫啉以及4%阿维·啶虫脒对牛角花齿蓟马和豌豆蚜均有较高的防效，最高防效均达到85.00%以上，但这2种药剂对传粉昆虫威胁较大，而0.3%印楝素则对传粉昆虫威胁较小。推荐花期苜蓿种子田使用0.3%印楝素1 000倍稀释液防控害虫，而非开花期可交替使用70%吡虫啉1 000倍稀释液和4%阿维·啶虫脒1 000倍稀释液防控害虫。     

Mycorrhizal protection of chili plants challenged by <Emphasis Type="Italic">Phytophthora capsici</Emphasis>

Hydrogen peroxide (H₂O₂) has been implicated in many stress conditions. Control of H₂O₂ levels is complex and dissection of mechanisms generating and relieving H₂O₂ stress is difficult, particularly in intact plants. Here the role of the mycorrhizal inoculation in chili plants challenged with Phytophthora capsici was investigated to study the effect on hypersensitive response. In the treatment without mycorrhiza (treatment T3) and with mycorrhiza (considered treatment T4) visible disorders were detected two days after inoculation with P. capsici, but in the next days T3 plants rapidly developed 25% more necrotic lesions on the leaves than T4 plants. Leaf necrosis correlated with H₂O₂ accumulation and the greater damage observed in T3 plants coincided with larger accumulation of H₂O₂ after 12 h of inoculation accompanied with an increase in POX (peroxidase) and SOD (superoxide dismutase) activity. T4-infected and mycorrhizal plants exhibited an earlier accumulation of H₂O₂ starting 6 h after inoculation with lower levels compared to T3 plants. Correlated with observed damage, POX and SOD activity measured in T4 plants indirectly suggest a smaller accumulation of ROS (reactive oxygen species) leading to a decrease in the wounds observed and slightly diminishing the advance of the pathogen. According to these findings, we conclude that mycorrhizal colonization contributes significantly in maintaining the redox balance during oxidative stress, but the exact mechanism is still uncertain.     

DNA sequence analysis of ribosomal ITS region 1 of Phytophthora vignae f. sp. adzukicola, the pathogen that causes stem rot of adzuki bean

Sequences of the internal transcribed spacer (ITS) region 1 were used to examine the phylogenetic relationships among races of 19 isolates of Phytophthora vignae f. sp. adzukicola and between this forma specialis and three isolates of the closely related P. vignae f. sp. vignae. The ITS 1 sequences were highly conserved (> 98.7% similarity) among representatives of both formae speciales groups. The results of this study indicate that P. vignae is a monophyletic group. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession nos. AB120062–AB120080 and AB120122     

Internal fruit rot of netted melon caused by <Emphasis Type="Italic">Pantoea ananatis</Emphasis> (=<Emphasis Type="Italic">Erwinia ananas</Emphasis>) in Japan

An internal fruit rot with a malodor was found in netted melons (Cucumis melo L.) in commercial greenhouses in Kochi Prefecture, Japan, in 1998, despite their healthy appearance and lack of water-soaking or brown spots on the surface. A yellow bacterium was consistently isolated from the affected fruits. To confirm the pathogenicity of eight representative isolates of the yellow bacterium, we stub-inoculated ovaries (immature-fruits) 5–7 days after artificial pollination, with a pin smeared with bacteria. After the melon fruits had grown for 60 more days, an internal fruit rot resembling the natural infection appeared, and the inoculated bacterium was reisolated. The melon isolates had properties identical with Pantoea ananatis, such as gram-negative staining, facultative anaerobic growth, indole production, phenylalanine deaminase absence, and acid production from melibiose, sorbitol, glycerol, and inositol. Phylogenetic analysis based on 16S rDNA sequences showed that the melon bacterium positioned closely with known P. ananatis strains. The melon bacterium had indole acetic acid (IAA) biosynthesis genes (iaaM and iaaH) and a cytokinin biosynthesis gene (etz). The bacterium could be distinguished from the other ‘Pantoea’ group strains by rep-PCR genomic fingerprinting. From these results, the causal agent of internal fruit rot was identified as a strain of P.ananatis [Serrano in (Philipp J Sci 36:271–305, 1928); Mergaert et al. in (Int J Syst Bacteriol 43:162–173, 1993)]. The nucleotide sequence data reported are available in the DDBJ database under accessions AB297969, AB373739, AB373740, AB373741, AB373742, AB373743 and AB373744.