The role of ethylene and wound signaling in resistance of tomato to Botrytis cinerea

Ethylene, jasmonate, and salicylate play important roles in plant defense responses to pathogens. To investigate the contributions of these compounds in resistance of tomato (Lycopersicon esculentum) to the fungal pathogen Botrytis cinerea, three types of experiments were conducted: (a) quantitative disease assays with plants pretreated with ethylene, inhibitors of ethylene perception, or salicylate; (b) quantitative disease assays with mutants or transgenes affected in the production of or the response to either ethylene or jasmonate; and (c) expression analysis of defense-related genes before and after inoculation of plants with B. cinerea. Plants pretreated with ethylene showed a decreased susceptibility toward B. cinerea, whereas pretreatment with 1-methylcyclopropene, an inhibitor of ethylene perception, resulted in increased susceptibility. Ethylene pretreatment induced expression of several pathogenesis-related protein genes before B. cinerea infection. Proteinase inhibitor I expression was repressed by ethylene and induced by 1-methylcyclopropene. Ethylene also induced resistance in the mutant Never ripe. RNA analysis showed that Never ripe retained some ethylene sensitivity. The mutant Epinastic, constitutively activated in a subset of ethylene responses, and a transgenic line producing negligible ethylene were also tested. The results confirmed that ethylene responses are important for resistance of tomato to B. cinerea. The mutant Defenseless, impaired in jasmonate biosynthesis, showed increased susceptibility to B. cinerea. A transgenic line with reduced prosystemin expression showed similar susceptibility as Defenseless, whereas a prosystemin-overexpressing transgene was highly resistant. Ethylene and wound signaling acted independently on resistance. Salicylate and ethylene acted synergistically on defense gene expression, but antagonistically on resistance.     

Nitric oxide negatively modulates wound signaling in tomato plants

Synthesis of proteinase inhibitor I protein in response to wounding in leaves of excised tomato (Lycopersicon esculentum) plants was inhibited by NO donors sodium nitroprusside and S-nitroso-N-acetyl-penicillamine. The inhibition was reversed by supplying the plants with the NO scavenger 2-(4-carboxiphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. NO also blocked the hydrogen peroxide (H(2)O(2)) production and proteinase inhibitor synthesis that was induced by systemin, oligouronides, and jasmonic acid (JA). However, H(2)O(2) generated by glucose oxidase and glucose was not blocked by NO, nor was H(2)O(2)-induced proteinase inhibitor synthesis. Although the expression of proteinase inhibitor genes in response to JA was inhibited by NO, the expression of wound signaling-associated genes was not. The inhibition of wound-inducible H(2)O(2) generation and proteinase inhibitor gene expression by NO was not due to an increase in salicylic acid, which is known to inhibit the octadecanoid pathway. Instead, NO appears to be interacting directly with the signaling pathway downstream from JA synthesis, upstream of H(2)O(2) synthesis. The results suggest that NO may have a role in down-regulating the expression of wound-inducible defense genes during pathogenesis.     

Nitric oxide and hydrogen peroxide: two players in the defence response of tomato plants to root-knot nematodes

Nitric oxide (NO) has been postulated to be required, together with reactive oxygen species (ROS) for activation of disease resistance reaction of plants to pathogen infection. Here, we investigated NO, superoxide (O(*-)2), and hydrogen peroxide (H2O2) in tomato-root-knot nematode interactions to answer the question of whether they are produced during the early stages of nematode infection. NO detection was carried out using diaminofluorescein diacetate (DAF-2DA) by means of confocal laser microscopy and spectrophotometric analyses, and production of NO was estimated by monitoring the conversion of L-[U14C]arginine into L-[U14C]citrulline. O(*-)2 production was determined by using the tetrazolium salt, sodium,3'-{1-[phenylamino-carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT) and H2O2 was measured by using the Amplex Red H2O2/peroxidase assay. Results showed i) the highest NO production in tissues challenged by avr pathotype, 12h after nematode inoculation, ii) NO production by nitric oxide synthase (NOS-like activity), iii) ROSbalance dependent control of NO. Our data evidenced, for the first time, that NO-generated signal, its spatiotemporal expression, and its cross-communication with other pro-oxidants or anti-oxidants critically influence compatible and incompatible tomato-Meloidogyne incognito interactions.     

Nitrogen availability and susceptibility of tomato leaves to Botrytis cinerea

The aim of this study was to investigate the effect of nitrogen availability on susceptibility of tomato leaves to the fungal pathogen Botrytis cinerea. Plants with varying nitrogen availability were grown by adding N daily in exponentially increasing amounts to a nutrient solution at different rates. Leaves of plants grown at low nitrogen availability had a high leaf C/N ratio (21 g g^-1) and were about 2.5 times more susceptible to primary lesion formation by B. cinerea compared to plant grown at high nitrogen availability, which had a low leaf C/N ratio (11 g g^-1). Leaf C/N ratio accounted for 95% of variation in susceptibility. This relationship between C/N ratio and susceptibility persisted when plants were grown with exponential P addition and optimal N supply, and was thus independent of plant growth rate or related factors. We could not explain the effect of nitrogen availability by variation in the most obvious N-based resistance compound α-tomatine because more susceptible leaves with a high C/N ratio contained more α-tomatine. These leaves also contained more soluble carbohydrates. The level of soluble carbohydrates correlated positively with susceptibility, independent of the growth method. We therefore suggest that the effect of N availability on susceptibility must be explained by variation in levels of soluble carbohydrates and speculate about the role of these carbohydrates in the infection process. This revised version was published online in June 2006 with corrections to the Cover Date.     

Functions of pipecolic acid on induced resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 in tomato plants

Amino acid metabolic pathways are involved in the plant immune system. Pipecolic acid (Pip), a lysine-derived non-protein amino acid, acts as an important regulator of disease resistance. Here, we report the functions of Pip on tomato disease resistance. Tomato seedlings treated with 0.5 mM Pip showed increased resistance to Pst DC3000 and B. cinerea compared with the control. After pathogen infection, the expression of defence-related genes increased in plants pretreated with Pip, while reactive oxygen species (ROS) accumulation decreased. These data demonstrated that exogenous application of Pip induced resistance against Pst DC3000 and B. cinerea in tomatoes, possibly through the regulation of ROS accumulation and defence-related gene expression.     

Nitric oxide modulates sensitivity to ABA

Nitric oxide (NO) is a gas with crucial signaling functions in plant defense and development. As demonstrated by generating a triple nia1nia2noa1-2 mutant with extremely low levels of NO (February 2010 issue of Plant Physiology), NO is synthesized in plants through mainly two different pathways involving nitrate reductase (NR/NIA) and NO Associated 1 (AtNOA1) proteins. Depletion of basal NO levels leads to a priming of ABA-triggered responses that causes hypersensitivity to this hormone and results in enhanced seed dormancy and decreased seed germination and seedling establishment in the triple mutant. NO produced under non-stressed conditions represses inhibition of seed developmental transitions by ABA. Moreover, NO plays a positive role in post-germinative vegetative development and also exerts a critical control of ABA-related functions on stomata closure. The triple nia1nia2noa1-2 mutant is hypersensitive to ABA in stomatal closure thus resulting in a extreme phenotype of resistance to drought. In the light of the recent discovery of PYR/PYL/RCAR as a family of potential ABA receptors, regulation of ABA sensitivity by NO may be exerted either directly on ABA receptors or on downstream signalling components; both two aspects that deserve our present and future attention.Key words: nitric oxide, abscisic acid, seed germination, stomata openingPlant development is the result of the succesfull execution of several programs that control the transition between different growth phases. Every developmental transition is regulated through coordinated mechanisms that involved exogenous environmental factors such as light and temperature as well as endogenous cues, including levels of primary and secondary metabolites. Among the latter, hormones such as gibberellins (GA), auxins, citokinins, ethylene and abscisic acid (ABA) participate in the control of most of the developmental transitions.^1,2 During the last years, nitric oxide (NO) has gained an increasing role as an essential player in plant defense responses³ as well as a co-regulator of many developmental processes.⁴ However, studies of NO function as a regulatory molecule in plants have been hampered by the scanty, limited and controversial knowledge on how this gas is synthesized in plants.^5,6 This situation has moved researchers in this area to adopt pharmacological approaches based on chemicals acting as artificial NO donors as well as inhibitors or scavengers of NO action. The lack of specificity and the inherent artificial effects of these chemicals can be overcome by genetic approaches based on the use of mutants with endogenous low levels of NO. In February 2010 issue of Plant Physiology, we report the generation and further characterization of a triple nia1nia2noa1-2 mutant that contains extremely low levels of NO due to the impairment of two NO biosynthetic pathways involving nitrate reductase (NIA/NR) or NO Associated 1 (AtNOA1) proteins.⁷ These findings support that NO is mainly produced through those pathways in Arabidopsis. However, the possible existence of a minor still uncharacterized pathways involved in the residual production of NO can not be ruled out at this time.Further functional characterization of nia1nia2noa1-2 mutant in terms of development has pointed to NO as an overall positive regulator of plant growth, affecting to almost every developmental stage from seed germination to reproductive development. Accordingly, triple mutant plants display a delayed growth resulting in small shoot and root size and they also produce low amounts of viable seeds.⁷Dormancy and seed germination are developmental programs largely regulated by the combined action of GA and ABA.¹ GA promote breaking of dormancy and promote germination whereas ABA acts as a brake in those processes, thus ensuring a timely seed germination. Our data from the characterization of dormancy and seed germination in the nia1nia2noa1-2 mutant suggest that NO’s role in the control of those processes may be exerted through modulation of the sensitivity to ABA (Fig. 1A). Seeds from NO deficient plants have increased dormancy and lower seed germination and seedling establishment rates than wild type seeds due to the enhanced ABA inhibitory action. These effects can be reversed by exogenous application of NO to nia1nia2noa1-2, suggesting that the sensitivity to ABA is actually controlled by the endogenous levels of NO. The recent identification of PYR/PYL/RCAR family of ABA receptors,^8,9 and the further characterization of the essential ABA regulatory module including receptor, protein phosphatases of the 2C class and kinases of the SnRK2 family¹⁰ point to these components as potential targets of NO in regulating sensitivity to ABA (Fig. 1B). This work is in progress in our lab but we already know that some of the PYR/PYL/RCAR receptors and SnRKs are actually regulated by NO and also that this regulation may be exerted at different levels (Lozano-Juste J and León J, unpublished data).Open in a separate windowFigure 1Interactions between NO and ABA results in modulated sensitivity to ABA throughout development. (A) NO synthesized through nitrate reductase (NR/NIA) and NO associatedI (AtNOA1) protein regulate germinative and post-germinative development as well as stomata movements through modulation of the sensitivity to ABA. Arrows and bars represent positive and negative effects, and the thickness of lines are proportional to the magnitud of regulatory effects. (B) Scheme of a minimal ABA signalling module and the potential targets of NO. Dashed lines represent effects still to be demonstrated. (C) ABA signalling in stomata guard cells through Ca²⁺-dependent and -independent pathways and the potential interactions with NO as represented by dashed lines.The enhanced sensitivity to ABA observed in germinative and post-germinative development of nia1nia2noa1-2, is extended throughout plant life cycle and it is actually the cause of the very strong resistance of nia1nia2noa1-2 plants to water deficit conditions.⁷ Stomatal aperture is a fine-tuned process controlled mainly through a balance between the light-promoted opening and the ABA-mediated promotion of closure and inhibition of opening¹¹ (Fig. 1A). It has been previously reported that ABA function on stomata movements involve the participation of NO as well as Ca²⁺ in such a way that Ca²⁺ chelators and NO scavengers block ABA action on stomata movements.¹² Stomata of nia1nia2noa1-2 leaves, despite of being depleted of NO, are not impaired for ABA inhibition of stomata opening but, in turn, they seem to be primed for a more efficient ABA response (Fig. 1A). Contrary to the Ca²⁺ requirements for ABA action on wild type stomata movements, this process is not affected by Ca²⁺ chelators in nia1nia2noa1-2 stomata, and it thus seems to be independent of Ca²⁺ in NO-deficient backgrounds (Fig. 1C). As mentioned above, NO might regulate sensitivity to ABA by acting on ABA receptors or on SnRKs, some of which are Ca²⁺-independent kinases. Both receptors and Ca^2+--independent kinases are likely targets of NO in the modulation of stomata sensitivity to ABA thus explaining the more efficient stomata closure in nia1nia2noa1-2 leaves, and the consequent low rates of evapotranspiration that leads to the extreme resistance of triple mutant plants to drought.The future characterization of the interactions between NO and key components of ABA signaling will be the basis for a better knowledge of the functional interactions between different hormones in plant development and defense, but it will also open up new possibilities of identifying new targets and strategies leading to improved drought resistance.     

Occurrence and management of fungicide resistance in Botrytis cinerea on tomato from greenhouses in Hebei,China

Grey mould, caused by the fungal pathogen Botrytis cinerea, is one of the most devastating tomato diseases, and the control of this disease is mainly by the application of chemicals. In this study, 512 isolates of B. cinerea were collected from tomato grown in greenhouses at 10 locations in 10 cities of Hebei Province from 2011 to 2016 and tested for their sensitivities to carbendazim (Car), diethofencarb (Die), iprodione (Ipr) and pyrimethanil (Pyr). Of these tested isolates, 95.7%, 95.2%, 31.6% and 89.4% were resistant to Car, Die, Ipr and Pyr, respectively. There were nine fungicide‐resistant phenotypes in the tested isolates. Car^RPyr^RDie^RIPR^S and Car^RPyr^RDie^RIPR^R were the most common phenotypes, accounting for 59.6%, and 31.1% of the tested isolates, respectively. The field trials showed that the control efficacies (CE) of carbendazim + diethofencarb (WP, 25% + 25%), pyrimethanil (EC, 40%) and iprodione (WP, 50%) at the recommended doses were 22.75%–29.23%, 58.44%–64.19% and 61.02%–65.17%, respectively, significantly lower than those of boscalid (WG, 50%) and pyrisoxazole (EC, 25%). The resistance management trial conducted from 2015 to 2017 indicated that the CE of tomato grey mould in the experimental fields was higher than 90% and the sensitivity to carbendazim, diethofencarb and pyrimethanil of B. cinerea isolates from the experimental fields increased on a yearly basis. These results showed that the frequency of resistance to Car, Die, Ipr and Pyr was high, and these four fungicides could not effectively control tomato grey mould. Tomato grey mould could be controlled by using biopesticides and newly synthesized fungicides with different modes of action. Our findings would be useful in designing and implementing fungicide resistance management spray programmes for the control of tomato grey mould.     

Benomyl tolerance in isolates of Botrytis cinerea from tomato plants

Three hundred and forty-nine isolates of Botrytis cinerea were collected from tomato crops on forty-one nurseries and 173 (40/6 %) were found to be tolerant to benomyl. There was no obvious association between disease incidence and the occurrence of tolerance. In a fungicide comparison experiment on tomatoes in 1973, twenty of the sixty-four (31 %) isolates examined were benomyl tolerant, the majority of these were from benomyl sprayed plants. In 1974 in a similar experiment, 384 of the 394 (97-5 %) isolates examined were tolerant. Tolerance was monitored in two tomato experiments in relation to a spray programme in which benomyl and dichlofluanid were used in various combinations. There was no marked effect of the spray programmes on the incidence of tolerance on either site. In the experiments B. cinerea was controlled and significant increases in yield were obtained with benomyl in 1973 but not in 1974. This difference is attributed to the change in the pathogen population with a large increase in the incidence of tolerance on the experimental site in 1974.     

不同寄主来源的灰葡萄孢对番茄的致病力分化研究

Eighteen isolates of Botrytis cinerea were obtained from the diseased plant tissue collected in Hefei, Bengbu, Changfeng and Hexian in Anhui province, by means of tissue isolating method. The pathogenicity of the isolates of B. cinerea from different hosts to the fruits and leaves of tomato were investigated by applying wound inoculation with mycelial blocks. The results showed that all of the tested isolates caused grey mould on tomato fruits, but there was significant difference in the average diameters of the lesions caused by different isolates, suggesting that there was significant differentiation in pathogenicity of B. cinerea strains to tomato fruits among isolates. According to the average diameters of the lesions on tomato fruits, the pathogenicity of the all isolates was classified into three categories: strong, intermediate and weak. In general, the isolates from tomato were more strongly pathogenic to tomato fruits than the isolates from strawberry, grape and capsicum. However, there was difference in pathogenicity among the different isolates from the same host, and the pathogenicity difference was not obviously related to the localities of isolates. After inoculating of tomato leaves, all of the tested isolates except CF3 caused grey mould on tomato leaves, but there was significant difference in the average diameters of the lesions caused by different isolates; and the difference in pathogenicity to tomato leaves was not obviously related to the host and locality of isolates.     

不同寄主来源的灰葡萄孢对番茄的致病力分化研究

从安徽合肥、蚌埠、长丰、和县等市、县的番茄、辣椒、草莓、葡萄等发病寄主上分离鉴定获得18个灰葡萄孢Botrytis cinerea菌株,采用菌丝块创伤接种法,分别测定了上述不同寄主来源的灰葡萄孢菌对番茄果实和叶片的致病力.结果表明,所有供试菌株接种番茄果实后均引起发病,但不同菌株所致病斑的平均直径有显著差异,显示灰葡萄孢菌株间对番茄果实的致病力存在明显分化.按照在番茄果实上所致病斑的平均直径大小可将供试菌株致病力划分为较强、中等和较弱3种类型.总体来说,来自番茄的菌株对番茄果实的致病力较强,来自草莓、葡萄和辣椒的菌株对番茄果实的致病力较弱,但来自相同寄主的菌株间致病力也存在差异,菌株致病力差异与菌株地域来源无明显相关.供试灰葡萄孢菌株接种番茄叶片后,除CF1外,均可引起番茄叶片发病,但不同菌株所致番茄叶片病斑的平均直径也有显著差异;供试菌株对番茄叶片的致病力差异与菌株的寄主和地域来源无显著相关.     

Confocal microscopy study to understand Clonostachys rosea and Botrytis cinerea interactions in tomato plants

Clonostachys rosea, a biological control agent for plant diseases, is found in a variety of habitats and colonises and survives in different tissues. This antagonist is effective at controlling grey mould, which is caused by Botrytis cinerea, in different plant species. Despite the existing knowledge regarding the efficiency of C. rosea at biologically controlling grey mould, there are few studies concerning this interaction at the histological level. Therefore, we studied the antagonist–pathogen interactions using confocal microscopy. C. rosea survived in tomato tissues for at least 30 days between 18–30ºC. The antagonist colonised the wounded tomato stems faster and more efficiently than the pathogen. The colonisation of the leaf tissues by C. rosea was slow, and the spore concentration was poor in this experiment. Combined with the pathogen’s direct penetration into the leaves, this slow colonisation could cause the biological control to fail. C. rosea also preyed parasitically upon the pathogen’s hyphae, penetrated the tomato’s leaf tissue through the stomata and colonised the stem’s intercellular spaces. Root colonisation was abundant, with a dense hyphae network forming between epidermal cell junctions. This observation provided evidence that the fungus can penetrate via the roots. This paper will help to better define an application strategy for C. rosea in tomato propagation, with the goal of biological control or growth promotion, because to understand how the antagonist survives and interacts in its habitat will define how and when to apply it.     

Nitric oxide, superoxide, and hydrogen peroxide production in brain mitochondria after haloperidol treatment.

Inhibition of mitochondrial respiration and free radical induction have been suggested to be involved in haloperidol neurotoxicity. In this study, mice were injected i.p. with haloperidol, according to two different treatments: (a) a single injection (1 mg/kg), sacrificed 1 h after the injection (single-dose model); and (b) two injections (1 mg/kg each), sacrificed 24 h after the first dose (double-dose model). Determinations of oxygen consumption and hydrogen peroxide (H2O2) production rate were carried out in isolated brain mitochondria. Nitric oxide (NO) and superoxide (O2-) production rates were measured in submitochondrial particles (SMP). Single-dose haloperidol treatment produced a 33% inhibition in malate-glutamate-dependent respiration, while no significant changes were found after double-dose treatment. NO production was inhibited by 39 and 54% in SMP from haloperidol-treated mice (single- and double-dose treatments, respectively) (control value: 1.6 +/- 0.2 nmol/min mg protein). NO steady-state concentration was estimated at about 16.5 nM and was decreased by 40% by haloperidol treatment. Increases of 105 and 54% were found in succinate-supported O2- and H2O2 production rates, respectively, after haloperidol single-dose treatment. Haloperidol treatment generated a 248% increase in SMP O2- production rate when measured in the presence of NADH plus rotenone. Our results suggest that haloperidol neurotoxicity would be mediated by a decreased mitochondrial NO production, a decreased intramitochondrial NO steady-state concentration, and by an inhibition of mitochondrial electron transfer with enhancement of O2- and H2O2 production. This inhibition does not seem to be caused by increased NO or ONOO- formation.     

An autonomously replicating plasmid transforms Botrytis cinerea to phleomycin resistance

Abstract A transformation system has been developed for the pathogen fungus Botrytis cinerea , based on the utilization of the wide host plasmid pUT737 that contains the Sh ble gene, conferring resistance to phleomycin. Transformed protoplasts were regenerated at 10–25 μg ml⁻¹ of phleomycin, at a frequency of 25–40 transformants per μg of DNA, and they were resistant up to 50 μg ml⁻¹. Southern hybridization using undigested and digested total DNA showed the presence of circular autonomously replicating plasmid pUT737 in the transformants. Reisolated plasmid from transformed fungus transformed E. coli and rescued plasmid was identified as pUT737. Transformants were grown for four generations under non-selective conditions and replicative plasmids were still detected. Plasmids present in all transformants at this stage had been modified from native pUT737 and showed the same size and configuration indicating that selection through stabilizing plasmid forms has happened.     

Factors affecting the resistance of cold-stored carrots to Botrytis cinerea

The secondary phloem parenchyma of cold-stored turgid roots of carrot (Daucus carota) is capable of localizing mycelial infection by Botrytis cinerea, producing a dark resistant lesion. The percentage of roots exhibiting this reaction declined with increasing time in cold-store: when freshly harvested and wound-inoculated in October 1974, 99%of roots resisted invasion, whereas only 5% of those stored until March 1975 did so. The surface dimensions of resistant lesions did not increase between 33 and 55 days after inoculation. However, the surface dimensions and mean weights of lesions (arising from inoculations performed at different times over the course of the storage season) were both larger with increasing time in storage of roots prior to inoculation. The ability of the root tissue to localize infection was reduced if the roots lost 5–10% or more of their fresh weight before inoculation, resulting progressively in susceptibility. Compared with roots wound-inoculated using mycelial disks, there was an overall reduction in infection when carrots were wound-inoculated using conidia or when conidia or mycelial disks were inoculated onto the apparently undamaged surface of roots.