Tenuazonic acid,toxin of rice blast fungus,induces disease resistance and reactive oxygen production in plants

Effects of tenuazonic acid (TA) on rice leaf segments and on their interaction with compatible races of the blast fungus (Magnaporthe grisea, former name is Pyricularia oryzae) were studied. TA induced small brown necrotic spots on leaves Application of TA (1 or 5 mM) to leaves in mixtures with M. grisea spores induced a local disease resistance, which reduced the frequency of compatible lesions. TA was not fungitoxic but, in contact with the leaf, increased the capability of leaf diffusates to inhibit germination of M. grisea spores. In the infected leaves, the diffusate fungitoxicity was higher than in the healthy ones. Antioxidant enzymes, superoxide dismutase and catalase, and scavengers of hydroxyl radical, mannitol and formate, strongly inhibited the TA-induced diffusate fungitoxicity. It is suggested that the disease resistance induced by TA is mediated, at least partially, by generation of reactive oxygen species by rice leaves, which inhibit the development of the fungus directly or indirectly.     

Role of hydrogen peroxide and apoplastic peroxidase in tomato — Botrytis cinerea interaction

The aim of the research was to estimate the sensitivity of tomato tissue and spore from necrotrophic isolate of B. cinerea on H₂O₂. The influence of exogenic H₂O₂ and B. cinerea on plant tissue and on the activity of peroxidases (PO), catalase (CAT) and superoxide dismutase (SOD) in apoplastic tomato leaves fraction were investigated. It was proved that 40 mM H₂O₂ damaged the cells of a host, and inhibited in vitro germination of B.cinerea spores. Complete inhibition of germination was observed after the use 100 mM H₂O₂. In the presence of spores H₂O₂ was decomposed to H₂O and O₂. Trace activity of catalase was observed in a solution of spores used for inoculation. Necrosis which appeared on the leaves after 40 mM H₂O₂ treatment resembled hypersensitive response. On the leaves pretreated at this concentration the development of infection was observed. The H₂O₂ concentration harmful for the tissues, stimulated the PO activity measured with NADH — responsible for generation of ^·O ₂ ⁻ , as well as with syringaldazine (S) and ferulic acid (FA), substrates characteristics of forms lignifying and strengthening the cell wall. Clear increase in CAT activity, resulting from infection and early pretreatment with H₂O₂ was observed in apoplast. No effect on SOD activity was observed. A hypothesis may be put forward, that germinating spores produce enzymes which allow them to decompose H₂O₂ generated in apoplast, so there is little likelihood that B. cinerea can be directly inhibited by reactive oxygen forms (ROS) during initial stages of infection. Necrotic lesions resembling HR generated by exogenous H₂O₂ as well as induction of activity of apoplastic plant enzymes, particularly PO connected with strengthening and lignification of cell wall, were not sufficient factors to inhibit fungal expansion.     

Exogenous superoxide dismutase may lose its antidotal ability on rice leaves

Leaf diffusates of the resistant rice (Oryza sativa L.) cultivars suppressed spore germination of blast fungus (Magnaporthe grisea (Hebert) Barr). Bovine Cu-Zn superoxide dismutase (SOD) added to the diffusate abolished its toxicity. However, the enzyme added to the inoculum did not affect the toxicity of the diffusate. Even the second SOD portion added to the diffusate was ineffective. As well, the enzyme exposed to leaves could not protect the fungus from artificially-generated superoxide. Presumably, SOD contacting with leaves induced an efflux of compound(s) inhibiting both portions of the enzyme. Evidence was obtained suggesting that neither enzymatic protein nor zinc of coenzyme but copper might be the inducer. A comparison of rice leaves and callus culture together with the effects of exogenous salicylic acid suggests that this compound may be the inhibitor liberating from leaves. It is not excluded that rice plants are capable of inactivation of antioxidant enzymes of pathogens and that this ability favors disease resistance.     

Salicylic and Jasmonic acids in regulation of the proantioxidant state in wheat leaves infected by <Emphasis Type="Italic">Septoria nodorum</Emphasis> Berk

Influence of mediators of the signal systems of salicylic (SA) and jasmonic (JA) acids and their mixture on reactive oxygen species’ (ROS) (superoxide radical and O₂^·− H₂O₂) generation and activity of oxidoreductases (oxalate oxidase, peroxidase and catalase) in leaves of wheat Triticum aestivum L. infected by Septoria leaf blotch pathogen Septoria nodorum Berk has been studied. Presowing treatment of seeds by SA and JA decreased the development rate of fungus on wheat leaves. SA provided earlier inductive effect on production of O₂^·− and H₂O₂ compared with JA. The protective effect of the salicylic and jasmonic acids against Septoria leaf blotch pathogen was caused by activation of oxalate oxidase, induction of anion and cation peroxidases, and decrease of catalase activity. Ability of compounds to stimulate ROS in the plant tissues can be used as criteria for evaluation of immune-modulating activity of new substances for protection of the plants.     

Rice resistance to blast caused by leaf surface moistening prior to inoculation

Effect of water droplets placed onto rice (Oryza sativa L.) leaves before inoculation with blast fungus Magnaporthe grisea (Hebert) Barr on disease severity and superoxide radical generation by the leaves was investigated. The leaves were inoculated by placement of spore suspension droplets. One day before, droplets of distilled water were placed to the same sites as an inoculum. It was found, that such a pretreatment decreased frequency of susceptible-type lesions by 2 to 2.5 times and increased that of symptomless outcome by 1.5 times in comparison with the nontreated control. Besides, the pretreatment enhanced superoxide radical generation in diffusates of healthy leaves of susceptible cultivar and in diffusates of infected leaves of resistant cultivar one day post inoculation. It is suggested that water contacting with the leaf surface for a rather long time washes out from its cells compounds possessing properties of plant endogenous elicitors. The latter induce superoxide radical formation by plants and, as a consequence, their disease resistance. This may be interpreted as plant adaptation to high humidity, which usually favors infections.     

Effect of ozone on spore germination,spore production and biomass production in two <Emphasis Type="Italic">Aspergillus</Emphasis> species

The ability of ozone gas to reduce food spoilage is relatively well documented, but the developmental effects of the gas on food spoilage fungi are not well known. In this study two model aspergilli, Aspergillus nidulans and Aspergillus ochraceus were used to study the effects of ozone on spore germination, sporulation and biomass production. These effects were examined under three levels of ozone; two high level ozone exposures (200 and 300 μmol mol⁻¹) and one low level exposure (0.2 μmol mol⁻¹). The two species behaved noticeably different to each other. Ozone was more effective in reducing growth from spore inocula than mycelia. No spore production could be detected in A. nidulans exposed to continuous low level O₃, whereas the same treatment reduced spores produced in A. ochraceus by 94%. Overall the work suggests that ozone exposure is an effective method to prevent spread of fungal spores in a food storage situation.     

Effects of the sporulation conditions on the lovastatin production by Aspergillus terreus

The production of biomass and lovastatin by spore-initiated submerged fermentations of Aspergillus terreus ATCC 20542 was shown to depend on the age of the spores used for inoculation. Cultures started from older spores produced significantly higher titers of lovastatin. For example, the lovastatin titer increased by 52% when the spore age at inoculation rose from 9 to 16 days. The lovastatin titer for a spore age of 16 days was 186.5±20.1 mg L⁻¹. The time to sporulation on surface cultures was sensitive to the light exposure history of the fungus and the spore inoculation concentration levels. A light exposure level of 140 μE m⁻² s⁻¹ and a spore concentration of 1,320 spore cm⁻² produced the greatest extent of sporulation within about 50 h of inoculation. Sporulation was slowed in the dark and with diluted inoculants. A rigorous analysis of the data of statistically designed experiments showed the above observations to be highly reproducible.     

Involvement of reactive oxygen species during early stages of ectomycorrhiza establishment between <Emphasis Type="Italic">Castanea sativa</Emphasis> and <Emphasis Type="Italic">Pisolithus tinctorius</Emphasis>

Evidence for the participation of reactive oxygen species (ROS) and antioxidant systems in ectomycorrhizal (ECM) establishment is lacking. In this paper, we evaluated ROS production and the activities of superoxide dismutase (SOD) and catalase (CAT) during the early contact of the ECM fungus Pisolithus tinctorius with the roots of Castanea sativa (chestnut tree). Roots were placed in contact with P. tinctorius mycelia, and ROS production was evaluated by determining the levels of H₂O₂ and O₂ ^·− during the early stages of fungal contact. Three peaks of H₂O₂ production were detected, the first two coinciding with O₂ ^·− bursts. The first H₂O₂ production peak coincided with an increase in SOD activity, whereas CAT activity seemed to be implicated in H₂O₂ scavenging. P. tinctorius growth was evaluated in the presence of P. tinctorius-elicited C. sativa crude extracts prepared during the early stages of fungal contact. Differential hyphal growth that matched the H₂O₂ production profile with a delay was detected. The result suggests that during the early stages of ECM establishment, H₂O₂ results from an inhibition of ROS-scavenging enzymes and plays a role in signalling during symbiotic establishment.     

Cadmium-induced early changes in O2 •−, H2O2 and antioxidative enzymes in soybean (Glycine max L.) leaves

Cadmium-induced initial changes in the production of reactive oxygen species (ROS) and antioxidant mechanism were investigated in soybean (Glycine max L. cv. Don Mario 4800 RR) leaves. Whole plants (WP) and plants without roots (PWR) were exposed to 0.0, 10.0 and 40.0 μM Cd for 0, 4, 6 and 24 h. Compared to PWR, a higher level of endogenous Cd in WP was associated with a lower oxidative stress measured in terms of lipid peroxidation. Furthermore, O₂ ^•− content decreased in the leaves of Cd-treated WP, whereas it increased in those of Cd-treated PWR. Although O₂ ^•− accumulation in PWR was associated with a decrease in superoxide dismutase (SOD) activity, O₂ ^•− diminution in WP leaves was not related to any increase in SOD activity. H₂O₂ content increased in the leaves of both Cd-treated WP and PWR, and it was concomitant with a corresponding decline in catalase (CAT) and ascorbate peroxidase (APX) activities. When diphenyl iodonium (DPI), an inhibitor of NADPH oxidase, was added, H₂O₂ content remained unchanged in Cd-treated WP, suggesting that NADPH oxidase does not participate in the early hours of Cd toxicity. Taken together, our results showed that early ROS evolution and oxidative damage were different in WP and PWR. This suggests that the response in soybean leaves during the early hours of Cd toxicity is probably modulated by the root.     

Effects of reactive oxygen species on α-tocopherol production in mitochondria and chloroplasts of Euglena gracilis

The effects of reactive oxygen species (ROS) on α-tocopherol production in mitochondria and chloroplasts of Euglena gracilis were investigated. Addition of an organic carbon source to the medium resulted in increased mitochondrial activity, intracellular O₂ ^- concentration and α-tocopherol productivity in E. gracilis W₁₄ZUL (a chloroplast deficient mutant). α-Tocopherol productivity of the wild-type strain (with both mitochondria and chloroplast) was higher than that of the W₁₄ZUL strain. In the case of the wild strain, the O₂ ⁻ generated in chloroplasts was efficiently scavenged by the α-tocopherol synthesized inside the chloroplast. In photoheterotrophic culture (with an organic carbon source), there was a positive correlation between α-tocopherol production and O₂ ⁻ generation. Addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (an inhibitor of photosynthesis) resulted in increased O₂ ⁻ generation and α-tocopherol productivity. These results indicate that the ROS generated in mitochondria and chloroplasts play important roles in α-tocopherol production by E. gracilis. The presence of chloroplasts and generation of intracellular ROS are important for efficient production of α-tocopherol.     

Molecular mapping of two cultivar-specific avirulence genes in the rice blast fungus <Emphasis Type="Italic">Magnaporthe grisea</Emphasis>

Rice blast, caused by the fungus Magnaporthe grisea, is a globally important disease of rice that causes annual yield losses. The segregation of genes controlling the virulence of M. grisea on rice was studied to establish the genetic basis of cultivar specificity in the interaction of rice and M. grisea. The segregation of avirulence and virulence was studied in 87 M. grisea F₁ progeny isolates from a cross of two isolates, Guy11 and JS153, using resistance-gene-differential rice cultivars. The segregation ratio indicated that avirulence and virulence in the rice cultivars Aichi–asahi and K59, respectively, are controlled by single major genes. Genetic analyses of backcrosses and full-sib crosses in these populations were also performed. The χ² test of goodness-of-fitness for a 1:1 ratio indicated that one dominant gene controls avirulence in Aichi-asahi and K59 in this population. Based on the resistance reactions of rice differential lines harboring known resistance genes to the parental isolates, two genetically independent avirulence genes, AVR–Pit and AVR–Pia, were identified. Genetic linkage analysis showed that the SSR marker m355–356 is closely linked to AVR–Pit, on the telomere of chromosome 1 at a distance of approximately 2.3 cM. The RAPD marker S487, which was converted to a sequence-characterized amplified region (SCAR) marker, was found to be closely linked to AVR–Pia, on the chromosome 7 telomere at a distance of 3.5 cM. These molecular markers will facilitate the positional cloning of the two AVR genes, and can be applied to molecular-marker-assisted studies of M. grisea populations.     

Cadmium and zinc-mediated oxidative burst in tobacco BY-2 cell suspension cultures

Generation of reactive oxygen species (ROS) constitutes an important first reaction under many stress conditions in plants. We demonstrate that Nicotiana tabacum L. cv. Bright Yellow 2 (TBY-2) cells in suspension cultures, generate superoxide radical and hydrogen peroxide upon treatment with cadmium and zinc. Addition of catalase and N,N-diethyldithiocarbamate (DDC) decreased the level of H₂O₂, whereas superoxide dismutase (SOD) induced a slight increase of the H₂O₂ production. The effects of catalase, DDC and SOD on the heavy metal-induced ROS production indicate that it occurs outside of the cells, and that at least part of the hydrogen peroxide is produced by dismutation of the superoxide radical (O₂ ^·−). The effect of pretreatment of the cell cultures with commonly used mammalian NADPH oxidase inhibitors was also tested. Strong inhibitions of cadmium and zinc-mediated ROS production were obtained with the flavoprotein inhibitors—diphenylene iodonium (DPI) and quinacrine and with an inhibitor of b-type cytochromes—imidazol. Membrane permeable-N-ethyl maleimide (NEM) and iodoacetate, and membrane non-permeable thiol reagents—para-chloromercuribenzoic acid (pCMBS) also inhibited the ROS production. These results suggested that the enzyme responsible for cadmium and zinc-induced ROS production in tobacco cells contains a flavocytochrome. They also show the importance of intra- and extracellular thiol groups in the observed stress reaction. The induction of ROS production with heavy metals showed properties comparable to the elicitor-induced oxidative burst in other plant cells.     

Autonomous and non-autonomous traits mediate social cooperation in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

In the trishanku (triA ⁻ ) mutant of the social amoeba Dictyostelium discoideum, aggregates are smaller than usual and the spore mass is located mid-way up the stalk, not at the apex. We have monitored aggregate territory size, spore allocation and fruiting body morphology in chimaeric groups of (quasi-wild-type) Ax2 and triA ⁻ cells. Developmental canalisation breaks down in chimaeras and leads to an increase in phenotypic variation. A minority of triA ⁻ cells causes largely Ax2 aggregation streams to break up; the effect is not due to the counting factor. Most chimaeric fruiting bodies resemble those of Ax2 or triA ⁻ . Others are double-deckers with a single stalk and two spore masses, one each at the terminus and midway along the stalk. The relative number of spores belonging to the two genotypes depends both on the mixing ratio and on the fruiting body morphology. In double-deckers formed from 1:1 chimaeras, the upper spore mass has more Ax2 spores, and the lower spore mass more triA ⁻ spores, than expected. Thus, the traits under study depend partly on the cells’ own genotype and partly on the phenotypes, and so genotypes, of other cells: they are both autonomous and non-autonomous. These findings strengthen the parallels between multicellular development and behaviour in social groups. Besides that, they reinforce the point that a trait can be associated with a genotype only in a specified context.     

Physiopathological Effects of the NO Donor 3-Morpholinosydnonimine on Rat Cortical Synaptosomes

The NO donor 3-Morpholinosydnonimine (SIN-1) releases NO in the presence of molecular oxygen. In this study, we evaluated the effect of SIN-1 on mitochondria of rat cortical synaptosomes. We demonstrated in vitro that the amount of ONOO⁻ generated and H₂O₂ formation directly correlated with SIN-1 concentration. The mean oxygen consumption by synaptosomal mitochondria was approximately 3.8 nmol of O₂ min⁻¹ mg⁻¹ protein, which decreased significantly in the presence of SIN-1 1 mM to 2.5 nmol O₂ min⁻¹ mg⁻¹. This decrease was not modified by catalase or Trolox, demonstrating that ONOO⁻ was responsible for the effect. The same concentration of SIN-1 caused a significant decrease of ATP production by synaptosomal mitochondria and depolarized the mitochondrial membrane. Moreover, ROS production increased progressively and was completely inhibited by pre-incubation of synaptosomes with Trolox. Finally, phosphatidylserine was externalized and, at the same time, intrasynaptosomal lactate dehydrogenase decreased confirming both, the external membrane breakdown after the addition of SIN-1 and the damage to the synaptosomes.     

Reaction of winter oilseed rape callus to different concentrations of elicitors: pectinase or chitosan

The aim of the presented methodical experiments was 1) the evaluation if callus of winter oilseed rape (Brassica napus var. oleifera L.) initiates a defence reaction to fungal elicitors: pectinase (polygalacturonase) or chitosan, and 2) the choice of the elicitor doses, which evoke the strongest tissue reaction. The results obtained will be used in the next experiments relating the studies of pathogenesis mechanisms proceeding in rape plants infected by necrotrophic fungi. The defence response was estimated on the basis of changes in electrolyte leakage from cells, metabolic efficiency, phenolic content and catalase activity. In the experiment pectinase was used at concentration of 3, 8, 16, 133 and 166 μl per 1 cm³ of culture medium while chitosan at: 25, 50, 75 and 100 μg·cm⁻³. Both elicitors increased cell membrane permeability: pectinase at the doses equal or greater to 16 μl·cm⁻³ while chitosan of 25 μg·cm⁻³. The greatest metabolic inefficiency was observed in calli elicited with 16 μl·cm⁻³ pectinase and with chitosan of 100 μg·cm⁻³. The decrease in phenolic content was noted under influence of most doses of both elicitors. The highest catalase activity was evoked by pectinase of 8 μl·cm⁻³ and chitosan of 75 and 100 μg·cm⁻³. The results indicated that 8–16 μl·cm⁻³ of pectinase and 100 μg·cm⁻³ of chitosan caused the strongest defence reaction of oilseed rape tissue.     

Involvement of oxidative stress and role of antioxidative defense system in growing rice seedlings exposed to toxic concentrations of aluminum

When seedlings of rice (Oryza sativa L.) cultivar Pant-12 were raised in sand cultures containing 80 and 160 μM Al³⁺ in the medium for 5–20 days, a regular increase in Al³⁺ uptake with a concomitant decrease in the length of roots as well as shoots was observed. Al³⁺ treatment of 160 μM resulted in increased generation of superoxide anion (O₂ ⁻) and hydrogen peroxide (H₂O₂), elevated amount of malondialdehyde, soluble protein and oxidized glutathione and decline in the concentrations of thiols (-SH) and ascorbic acid. Among antioxidative enzymes, activities of superoxide dismutase (SOD EC 1.15.1.1), guaiacol peroxidase (Guaiacol POX EC 1.11.1.7), ascorbate peroxidase (APX EC 1.11.1.11), monodehydroascorbate reductase (MDHAR EC 1.6.5.4), dehydroascorbate reductase (EC 1.8.5.1) and glutathione reductase (EC 1.6.4.2) increased significantly, whereas the activities of catalase (EC EC 1.11.1.6) and chloroplastic APX declined in 160 μM Al³⁺ stressed seedlings as compared to control seedlings. The results suggest that Al³⁺ toxicity is associated with induction of oxidative stress in rice plants and among antioxidative enzymes SOD, Guaiacol POX and cytosolic APX appear to serve as important components of an antioxidative defense mechanism under Al³⁺ toxicity. PAGE analysis confirmed the increased activity as well as appearance of new isoenzymes of APX in Al³⁺ stressed seedlings. Immunoblot analysis revealed that changes in the activities of APX are due to changes in the amounts of enzyme protein. Similar findings were obtained when the experiments were repeated using another popular rice cv. Malviya-36.     

Aerobiological dynamics of potentially pathogenic fungi in a rice agroecosystem in La Habana, Cuba

The aerobiology can play a key role in protecting the rice crop since many fungi can cause serious damage to agricultural areas. In this way, the ideal time to implement different security measures can be identified. To determine the presence of potentially pathogenic fungi in the air of the rice agroecosystem, a weekly monitoring of viable fungi was carried out using a volumetric sampler. Collected fungi were quantified, isolated, and identified based on their morphological characteristics. The results obtained demonstrated that the annual average concentration of filamentous fungi in the atmosphere of rice agroecosystem studied was 1,225 cfu m⁻³ levels ranging between 115 cfu m⁻³ (April) and 2,865 cfu m⁻³ (August). Pyricularia grisea was detected in the air for 5 months, since the second week of June until the first week of October, and highest average concentration (25 cfu m⁻³) was observed in August. Of the meteorological factors evaluated, temperature and relative air humidity influence the concentration of propagules of P. grisea in the air. Besides, other fungi were detected such as Curvularia, Bipolaris, Alternaria, and Cercospora, all with relevance to rice cultivation. This is the first characterization of aeromycological biodiversity in the studied region.     

Photosynthetic and yield responses of an old and a modern winter wheat cultivars to short-term ozone exposure

In order to study the responses of winter wheat cultivars released in different years to short-term high O₃ exposure, an old cultivar (‘Nongda 311’, released in 1960s) and a modern one (‘Yannong 19’, released in 1990s) were treated with an O₃ exposure (145 ± 12 mm³ m⁻³, 4 h d⁻¹ for 3 d) shortly after anthesis stage (> 50 % main stems blossomed). During the O₃ exposure, light-saturated photosynthetic rate (P _N) and stomatal conductance (g _s) of both cultivars decreased considerably. Elevated O₃ did not decrease dark-adapted maximum photochemical efficiency, but induced significant reduction in actual photochemical efficiency and thereby considerably increase in non-photochemical quenching. P _N, g _s of the modern cultivar ‘Yannong 19’ decreased more than the older one ‘Nongda 311’, indicating the former exhibited higher sensitivity to O₃ than the latter. After O₃ exposure, P _N, g _s and chlorophyll (Chl) content in flag leaf decreased more quickly than control, indicating induction of faster premature leaf senescence. As a result, the short-term O₃ exposure caused substantial yield loss, with larger reduction in ‘Yannong 19’ (−19.2 %) than in ‘Nongda 311’ (−8.4 %). Our results indicated that high O₃ exposure at grain filling stage would have greater negative impacts on the high yielding modern cultivar relative to the old one with lower yield.     

Killing bacterial spores by organic hydroperoxides

Killing of wild-type spores of Bacillus subtilis by t-butyl hydroperoxide, cumene hydroperoxide and peracetic acid was not through DNA damage, as shown by the absence of mutations in the survivors and the identical sensitivity of spores of strains with or without a recA mutation. In contrast, B. subtilis spores (termed α⁻β⁻) lacking the DNA protective α/β-type small, acid-soluble spore proteins (SASP) were more sensitive to t-butyl hydroperoxide and cumene hydroperoxide, and their killing was in large part through DNA damage, as shown by the high frequency of mutations in the survivors and the greater sensitivity of α⁻β⁻ recA spores. Analysis of t-butyl hydroperoxide-treated spores showed that generation of DNA damage in α⁻β⁻ spores was more rapid than in wild-type spores; α/β-type SASP also protected against DNA strand breakage in vitro caused by t-butyl hydroperoxide. α/β-Type SASP appeared to play no role in protection of spores from killing by peracetic acid; wild-type and α⁻β⁻ spores exhibited identical peracetic acid sensitivity and their killing by this agent appeared to be not through DNA damage. Received 17 December 1996/ Accepted in revised form 13 March 1997