Nitric oxide and hydrogen peroxide in tomato resistance. Nitric oxide modulates hydrogen peroxide level in o-hydroxyethylorutin-induced resistance to Botrytis cinerea in tomato.

Nitric oxide (NO) has been postulated to be required, together with reactive oxygen species (ROS), for activation of disease resistance reactions of plants to infection with a pathogen or elicitor treatment. However, biochemical mechanisms by which ROS and NO participate in these reactions are still under intensive study and controversial debate. We previously demonstrated that o-hydroxyethylorutin when applied on tomato leaves (Lycopersicon esculentum Mill. cv. "Perkoz") restricted Botrytis cinerea infection development. In this research we investigated ROS and NO generation in tomato plants treated with o-hydroxyethylorutin, non-treated and infected ones. The NO content was enhanced or decreased in the studied plants by supplying them with NO generator-SNP or scavenger-cPTIO. NO detection was carried out using diaminofluorescein diacetate (DAF-DA) in conjunction with confocal laser scanning microscopy. The influence of elevated and decreased levels of NO on B. cinerea infection development and ROS generation was studied. The elevated NO concentration in tomato leaves strongly decreased hydrogen peroxide concentration without affecting other studied ROS (superoxide anion and hydroxyl radical) levels. H2O2 concentrations in NO-supplied leaves were low regardless of further treatment of tomato leaves with o-hydroxyethylorutin or inoculation with B. cinerea. The low H2O2 concentration coincided with quick and severe infection development in NO-supplied leaves. As activities of enzymes generating (SOD EC 1.15.1.1)) and removing (APX EC 1.11.1.11, CAT EC 1.11.1.6) H2O2 were unchanged in the studied plants, the decrease in H2O2 concentration was probably due to a direct NO-H2O2 interaction.     

Complementation of notabilis, an abscisic acid-deficient mutant of tomato: importance of sequence context and utility of partial complementation

The abscisic acid (ABA)‐deficient tomato mutant notabilis (not) is believed to be a null mutation in the gene LeNCED1, encoding a 9‐cis‐epoxycarotenoid dioxygenase involved in ABA biosynthesis. We have sequenced and analysed a 19 kb genomic clone containing LeNCED1 and 5.4 kb of its promoter. This clone was transferred to not homozygotes and several non‐wilty transformed plants were obtained. The basal ABA content, water relations, shoot and root growth, adventitious rooting, ethylene evolution and ability to accumulate ABA under water stress are described for two of these lines, not_comp.13 and not_comp.1. Partial complementation was observed for most parameters measured for not_comp.1. Full complementation was observed in not_comp.13 for all parameters measured in whole plants under well‐watered and water‐stressed conditions. These data provide further evidence that LeNCED1 is the wild‐type allele of the not mutant gene. However, not_comp.13 was unable to accumulate the wild‐type levels of ABA in rapidly dehydrated leaves, indicating that it too was only partially complemented. Since LeNCED1 is an environmentally regulated gene encoding a rate‐limiting enzyme, precise levels and patterns of gene expression may be needed to fully recreate wild‐type phenotype. The utility of partially complemented lines to study the role of ABA in plant responses to stress conditions, and in promoter analysis, is discussed.     

Identification of an abscisic acid gene cluster in the grey mold Botrytis cinerea

Like several other phytopathogenic fungi, the ascomycete Botrytis cinerea is known to produce the plant hormone abscisic acid (ABA) in axenic culture. Recently, bcaba1, the first fungal gene involved in ABA biosynthesis, was identified. Neighborhood analysis of bcaba1 revealed three further candidate genes of this pathway: a putative P450 monooxygenase-encoding gene (bcaba2), an open reading frame without significant similarities (bcaba3), and a gene probably coding for a short-chain dehydrogenase/reductase (bcaba4). Targeted inactivation of the genes proved the involvement of BcABA2 and BcABA3 in ABA biosynthesis and suggested a contribution of BcABA4. The close linkage of at least three ABA biosynthetic genes is strong evidence for the presence of an abscisic acid gene cluster in B. cinerea.     

番茄灰霉病拮抗内生细菌的筛选、鉴定及其活性

对安徽省淮北市番茄植株根、茎、叶中内生细菌及其数量进行了调查和筛选,并测定了其抑菌活性。结果表明,番茄根、茎和叶中的内生细菌的数量分别为5.69×105、5.16×105和2.83×105CFU.g-1鲜重。根据分离部位和表型特征,从健康番茄植株体内分离到267株内生细菌,通过对峙实验,筛选到11株对番茄灰霉病菌有拮抗作用的菌株,占所分离内生细菌总数的4.12%。来自茎组织中的菌株XF136的抑菌效果最佳,抑菌带宽度达32.2mm。根据形态特征、生理生化特性、(G+C)mol%和16SrDNA序列分析,将菌株XF136鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。室内测定菌株XF136发酵滤液对灰霉病菌菌丝生长及分生孢子萌发的抑制作用,结果表明,菌株XF136发酵滤液可以抑制灰霉病菌菌丝生长和分生孢子萌发,且浓度越高,抑制能力越强;当发酵滤液浓度为20%时则完全抑制灰霉病菌菌丝生长和分生孢子萌发。盆栽防效试验结果表明,10%菌株XF136发酵滤液对番茄灰霉病防治效果与50%多菌灵600倍液相当,20%发酵滤液对番茄灰霉病的防治效果高于50%多菌灵600倍液。本研究表明,菌株XF136是防治番茄灰霉病潜在的优良生防菌株,具有良好的开发应用价值。     

Growth rate, plant development and water relations of the ABA-deficient tomato mutant sitiens

Given the close relationship between a plant's growth rate and its pattern of biomass allocation and the effects of abscisic acid (ABA) on biomass allocation, we studied the influence of ABA on biomass allocation and growth rate of wildtype tomato ( Lycopersicon esculentum Mill. cv. Moneymaker) plants and their strongly ABA-deficient mutant sitiens. The relative growth rate of sitiens was 22% lower than that of the wildtype, as the result of a decreased specific leaf area. The net assimilation rate and the leaf weight ratio were not affected. The mutant showed a much higher transpiration rate and lower hydraulic conductance of the roots. These two factors resulted in sitiens having a significantly lower leaf water potential and turgor. resulting in reduced leaf expansion and, consequently, a lower specific leaf area relative to the wildtype. Addition of ABA to the sitiens roots resulted in phenotypic reversion to the wildtype. We conclude that the influence of ABA-deficiency on biomass allocation and relative growth rate is the result of altered water relations in the plants, rather than of a direct effect on sink strength of different plant organs.     

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.     

Resistance to Botrytis cinerea in canes of Rubus idaeus and some related species

Mycelial inoculation of canes with Botrytis cinerea proved a useful method for assessing Rubus material for resistance. When canes were inoculated in summer resistant genotypes developed relatively small lesions which produced few small sclerotia in the following spring. The size of lesions in autumn generally provided the best discrimination between genotypes. Very strong resistance was found in several species, of which Rubus pileatus and R. occidentalis are the most useful for breeding; hybrids of raspberry with these species or with R. crataegifolius also had strong resistance. The resistance shown by a derivative of red raspberry cv. Chief was less strong, as was the resistance conferred by gene H, which determines cane pubescence.     

Compartment-specific role of the ascorbate-glutathione cycle in the response of tomato leaf cells to Botrytis cinerea infection

Changes in AA-GSH cycle activity following Botrytis cinerea infection were studied in tomato whole-leaf extracts as well as in chloroplasts, mitochondria, and peroxisomes. The oxidative effect of infection affected all cellular compartments although mitochondria and peroxisomes underwent the most pronounced changes. Apart from organelle-specific variations, a general shift of the cellular redox balance towards the oxidative state was found. It was manifested by the significant decline in concentrations and redox ratios of the ascorbate and glutathione pools as well as by the insufficient activity of MDHAR, DHAR, and GR needed for antioxidant regeneration. There was no compatibility between the ascorbate- and glutathione-mediated changes in different compartments. It was concluded that B. cinerea was able to break down the protective antioxidant barrier of the AA-GSH cycle at both the cellular and organellar levels. The changes in the AA-GSH cycle activity could partly be related to the B. cinerea-induced promotion of senescence that favoured disease progress.     

Effect of UV-C on Phytoalexin Accumulation and Resistance to Botrytis cinerea in Stored Carrots

The effect of UV-C (220–280 nm) on the accumulation of phytoalexin and resistance to Botrytis cinerea was studied in cold-stored carrots. Carrots were surface-wounded, treated with a range of UV doses and stored at 1 °C for 25 days in lots of 22 roots. The level of the phytoalexin, 6-methoxymellein, in each lot was then assayed in the peel of eight roots. Twelve of the remaining roots were subsequently inoculated with mycelial plugs to evaluate their level of disease resistance. The elicitation of 6-methoxymellcin by UV increased significantly the resistance of the roots to B. cinerea. The effect of UV in freshly harvested carrots was curvilinear, showing an optimum between 0.44 and 0.88 Merg/cm². However, only a linear relationship was observed with aged (stored for 4 months at 1 °C) carrots for the same doses, suggesting a modification in the response to UV with age. Wounding was necessary for carrots kept at 1 °C to respond to UV treatment. Neither UV nor wounding alone caused any elicitation at this temperature. Since unwounded roots could respond to UV at 20 °C, it is hypothesized that the level of physiological activity of the roots determines their response to UV. An increase in the physiological activity by higher temperatures or wounding would allow the elicitation process to take place. Since UV irradiation can increase the level of disease resistance in treated tissues, this treatment has potential as an alternative method for the control of post-harvest diseases m carrots.     

AtPLAI is an acyl hydrolase involved in basal jasmonic acid production and Arabidopsis resistance to Botrytis cinerea

Intracellular phospholipase A2 (PLA2) plays an important role in regulating oxylipin biosynthesis in mammals, but the molecular and biochemical nature of intracellular PLA2 is not well understood in plants. Arabidopsis thaliana gene At1g61850 (AtPLAI) encodes a 140-kDa protein that is most similar to mammalian calcium-independent PLA2, and additionally contains leucine-rich repeats and Armadillo repeats. AtPLAI hydrolyzes phospholipids at both the sn-1 and sn-2 positions, but prefers galactolipids to phospholipids as substrates. Profiling of lipid species altered in response to the necrotrophic fungus Botrytis cinerea revealed decreases in the levels of phosphatidylglycerol and digalactosyldiacylglycerol, suggesting that hydrolysis of plastidic polar lipids might provide precursors for pathogen-induced jasmonic acid (JA) production. Disruption of AtPLAI by T-DNA insertion reduced the basal level of JA, but did not impede pathogen-induced production of JA, free linolenic acid, or hydrolysis of plastidic lipids. Still, AtPLAI-deficient plants exhibited more damage than wild type plants after B. cinerea infection, and pretreatment of plants with methyl jasmonate alleviated pathogen damage to the mutant plants. The study shows that AtPLAI is an acyl hydrolase, rather than a specific phospholipase A. AtPLAI is involved in basal JA production and Arabidopsis resistance to the necrotrophic fungus B. cinerea.     

Loss-of-function mutation of REDUCED WALL ACETYLATION2 in Arabidopsis leads to reduced cell wall acetylation and increased resistance to Botrytis cinerea

Nearly all polysaccharides in plant cell walls are O-acetylated, including the various pectic polysaccharides and the hemicelluloses xylan, mannan, and xyloglucan. However, the enzymes involved in the polysaccharide acetylation have not been identified. While the role of polysaccharide acetylation in vivo is unclear, it is known to reduce biofuel yield from lignocellulosic biomass by the inhibition of microorganisms used for fermentation. We have analyzed four Arabidopsis (Arabidopsis thaliana) homologs of the protein Cas1p known to be involved in polysaccharide O-acetylation in Cryptococcus neoformans. Loss-of-function mutants in one of the genes, designated REDUCED WALL ACETYLATION2 (RWA2), had decreased levels of acetylated cell wall polymers. Cell wall material isolated from mutant leaves and treated with alkali released about 20% lower amounts of acetic acid when compared with the wild type. The same level of acetate deficiency was found in several pectic polymers and in xyloglucan. Thus, the rwa2 mutations affect different polymers to the same extent. There were no obvious morphological or growth differences observed between the wild type and rwa2 mutants. However, both alleles of rwa2 displayed increased tolerance toward the necrotrophic fungal pathogen Botrytis cinerea.     

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.     

Remote stimulation by heat induces characteristic membrane-potential responses in the veins of wild-type and abscisic acid-deficient tomato plants

The systemic induction of proteinase inhibitor genes in tomato plants is either mediated by fast electrical signals or alternatively by chemical messengers. In the present study we analyzed the pathway of the electrical signal. The question of which cell types are involved in this pathway of long-distance signaling within plants is still controversial. To identify these we inserted microelectrodes into the veins of tomato leaves (Lycopersicon esculentum Mill. cv. Moneymaker). A newly developed computer program and microcomputer interface enabled us to position these microelectrodes inside the vein with an accuracy of 1 μm. Due to this precision in positioning we were able to demonstrate that the pathway of the electrical signal is not restricted to a specific tissue type, e.g. the phloem. In particular, the entire vein contributes to the propagation of the electrical wave along the plant. Therefore, an apoplastic contribution to the long-distance signal transduction mechanism appears most likely. To furthermore investigate the involvement of cis-abscisic acid (ABA) in this long-distance signal transduction pathway, ABA-deficient tomato mutants (Lycopersicon esculentum cv. Sitiens) were used in comparison to the wild type. Significant differences between the membrane-potential relaxation kinetics of the wild type and the mutants could be detected. Wild-type tomato plants exhibited six characteristic classes of membrane-potential relaxation kinetics following heat treatment. In contrast, the ABA-deficient mutants were more restricted in terms of their relaxation upon heat stimulation. The responses in the membrane potential of all cells within a vein consisted of only three categories. In conclusion, ABA did not affect all cells within the vein in a similar manner. Single cells exhibited different response patterns to systemic heat application in the presence of ABA. Moreover, ABA had a pronounced effect on the resting potentials of individual cells within the veins of tomato. Received: 1 July 1997 / Accepted: 16 January 1998     

Absence of the endo-beta-1,4-glucanases Cel1 and Cel2 reduces susceptibility to Botrytis cinerea in tomato

Cel1 and Cel2 are members of the tomato (Solanum lycopersicum Mill) endo-beta-1,4-glucanase (EGase) family that may play a role in fruit ripening and organ abscission. This work demonstrates that Cel1 protein is present in other vegetative tissues and accumulates during leaf development. We recently reported the downregulation of both the Cel1 mRNA and protein upon fungal infection, suggesting the involvement of EGases in plant-pathogen interactions. This hypothesis was confirmed by assessing the resistance to Botrytis cinerea infection of transgenic plants expressing both genes in an antisense orientation (Anti-Cel1, Anti-Cel2 and Anti-Cel1-Cel2). The Anti-Cel1-Cel2 plants showed enhanced resistance to this fungal necrotroph. Microscopical analysis of infected leaves revealed that tomato plants accumulated pathogen-inducible callose within the expanding lesion. Anti-Cel1-Cel2 plants presented a faster and enhanced callose accumulation against B. cinerea than wild-type plants. The inhibitor 2-deoxy-d-glucose, a callose synthesis inhibitor, showed a direct relationship between faster callose accumulation and enhanced resistance to B. cinerea. EGase activity appears to negatively modulate callose deposition. The absence of both EGase genes was associated with changes in the expression of the pathogen-related genes PR1 and LoxD. Interestingly, Anti-Cel1-Cel2 plants were more susceptible to Pseudomonas syringae, displaying severe disease symptoms and enhanced bacterial growth relative to wild-type plants. Analysis of the involvement of Cel1 and Cel2 in the susceptibility to B. cinerea in fruits was done with the ripening-impaired mutants Never ripe (Nr) and Ripening inhibitor (rin). The data reported in this work support the idea that enzymes involved in cell wall metabolism play a role in susceptibility to pathogens.     

Pseudozyma aphidis activates reactive oxygen species production,programmed cell death and morphological alterations in the necrotrophic fungus Botrytis cinerea

Many types of yeast have been studied in the last few years as potential biocontrol agents against different phytopathogenic fungi. Their ability to control plant diseases is mainly through combined modes of action. Among them, antibiosis, competition for nutrients and niches, induction of systemic resistance in plants and mycoparasitism have been the most studied. In previous work, we have established that the epiphytic yeast Pseudozyma aphidis inhibits Botrytis cinerea through induced resistance and antibiosis. Here, we demonstrate that P. aphidis adheres to B. cinerea hyphae and competes with them for nutrients. We further show that the secreted antifungal compounds activate the production of reactive oxygen species and programmed cell death in B. cinerea mycelium. Finally, P. aphidis and its secreted compounds negatively affect B. cinerea hyphae, leading to morphological alterations, including hyphal curliness, vacuolization and branching, which presumably affects the colonization ability and infectivity of B. cinerea. This study demonstrates additional modes of action for P. aphidis and its antifungal compounds against the plant pathogen B. cinerea.     

Differences in the initial events of infection of Botrytis cinerea strains isolated from tomato and grape

Various stages of the infection process among B. cinerea strains isolated from tomatoes or grapes, belonging to different genetic groups, were compared. It was found that strains of B. cinerea isolated from either grapes or tomatoes showed differences in adhesion patterns and in the percentage of germination on tomato cutin. In strains isolated from tomato the first stage of adhesion occurred faster than in strains isolated from grape. At the same time strains isolated from tomato showed a higher percentage of germination on tomato cutin than the other strains after 9 h of incubation. The production and isoenzymatic patterns of polygalacturonases, pectin methyl esterases, pectin lyases, p-nitrophenylbutyrate esterases and laccases by B. cinerea in solid-state fermentation also were analyzed. Correlation between the production of these enzymes and the origin of the strains was not found. On the other hand all strains produced different isoenzymes and a common pattern between the strains was not observed. The ability of B. cinerea strains to colonize tomato leaves also differs between the isolated strains obtained from grapes and tomato. Strains isolated from tomato were more virulent on tomato leaves than strains isolated from grapes.     

拟南芥HyPRP蛋白AZI1在转基因烟草中的亚细胞定位及其对灰葡萄孢的抗性

通过农杆菌转化法得到了整合有拟南芥AZII基因的烟草植株,进一步利用转基因烟草分析了AZI1蛋白的亚细胞定位及其对真菌病原体的抗性特征。在上下游引物5’端分别引入NcoI和SpeI酶切位点,采用高保真耐热DNA聚合酶彤Pfu从拟南芥Co1-0生态型基因组DNA扩增AZII基因的编码序列,用NcoI和Spel对扩增片段和pCAMBIA1302质粒载体进行双酶切,通过T4DNA连接酶构建产生AZII-GFP融合表达载体。用包含融合表达载体的农杆菌细胞转化烟草叶片,经潮霉素选择获得了完整的再生植株,并收取了T。代种子。激光共聚焦显微观察发现,AZI1蛋白主要定位于细胞表面。病原体侵染结果显示,AZI1基因能够明显提高烟草对灰葡萄孢的抗性。说明AZI1蛋白通过分泌途径被定位到细胞表面后,能够抑制真菌病原体对植物组织的侵染过程。