Comparison of Antagonists of Botrytis cinerea in Greenhouse-grown Cucumber and Tomato under Different Climatic Conditions

The efficacy of Trichoderma harzianum T39 and the yeasts Aureobasidium pullulans and Cryptococcus albidus against Botrytis cinerea in cucumber and tomato was compared with chemical control. Four experiments were conducted in cucumber grown under different climatic conditions in The Netherlands, and two experiments were done in tomato both in the Netherlands and in Israel. T. harzianum and A. pullulans showed the most consistent control of B. cinerea, reducing stem lesions and death of plants by 40–100% in most cases. Control of stem lesions and subsequent wilting was generally better than control of symptoms on fruits. In some cases, the biocontrol agents were more effective than the broad-spectrum fungicide tolylfluanid and the selective fungicide iprodione. The climatic conditions did not strongly influence the efficacy of the biocontrol agents, but regression analysis showed that high temperature during the day and high vapour pressure deficit during the night reduced biocontrol efficacy. From the results, prospects for biocontrol of B. cinerea in greenhouse vegetables appear good under a range of conditions.     

Biological activity of the succinate dehydrogenase inhibitor fluopyram against Botrytis cinerea and fungal baseline sensitivity

BACKGROUND: Succinate dehydrogenase inhibitors (SDHIs) constitute a fungicide class with increasing relevance in crop protection. These fungicides could play a crucial role in successful management of grey mould disease. In the present study the effect of fluopyram, a novel SDHI fungicide, on several developmental stages of Botrytis cinerea was determined in vitro, and the protective and curative activity against the pathogen was determined on strawberry fruit. Furthermore, fungal baseline sensitivity was determined in a set of 192 pathogen isolates. RESULTS: Inhibition of germ tube elongation was found to be the most sensitive growth stage affected by fluopyram, while mycelial growth was found to be the least sensitive growth stage. Fluopyram provided excellent protective activity against B. cinerea when applied at 100 µg mL^?1 96, 48 or 24 h before the artificial inoculation of the strawberry fruit. Similarly, fluopyram showed a high curative activity when it was applied at 100 µg mL^?1 24 h post‐inoculation, but, when applications were conducted 48 or 96 h post‐inoculation, disease control efficacy was modest or low. The measurement of baseline sensitivity showed that it was unimodal in all the populations tested. The individual EC₅₀ values for fluopyram ranged from 0.03 to 0.29 µg mL^?1. In addition, no correlation was found between sensitivity to fluopyram and sensitivity to other fungicides, including cyprodinil, fenhexamid, fludioxonil, iprodione, boscalid and pyraclostrobin. CONCLUSIONS: The obtained biological activity, baseline sensitivity and cross‐resistance relationship data suggest that fluopyram could play a key role in grey mould management in the near future and encourage its introduction into spray programmes. Copyright © 2011 Society of Chemical Industry     

Flow of Botrytis cinerea inoculum between lettuce crop and soil

Botrytis cinerea causes grey mould, a disease common on many economically important crops. Although much attention is paid to the airborne inoculum of this fungus, as it sporulates abundantly in favourable conditions, knowledge on the abundance and genetic characteristics of soilborne inoculum could help improve control strategies. In this study, the soilborne inoculum of B. cinerea was quantified in two greenhouses at different times before and after the cultivation of four successive lettuce crops. Between 0 and 1177 colony‐forming units (CFU) of B. cinerea per gram of soil were recorded. There was no significant correlation between abundance of soilborne inoculum and subsequent disease incidence on lettuce (P = 0·11). Sixty‐five isolates collected from diseased plants and 66 isolates collected from the soil were investigated for their genetic diversity. The soil strains showed lower genetic diversity than the lettuce strains when considering the unbiased gene diversity within the nine microsatellite loci, the mean number of alleles per locus and the haplotypic diversity. The genetic differentiation between lettuce and soil strains decreased over three successive lettuce crops. At the same time, the genetic structure of the two groups of strains tended to become similar. These results are consistent with the hypothesis of a flow of inoculum between the lettuce crop and the soil, and vice versa. The study shows that grey mould management should pay more attention to the inoculum of B. cinerea present in the soil.     

Evaluation of strawberry (Fragaria L.) genetic resources for resistance to Botrytis cinerea

The grey mould disease caused by Botrytis cinerea leads to substantial economic losses in strawberry production all over the world. Control of the disease requires an extensive amount of fungicide that is applied in varying complexes because the pathogen easily develops resistance against the active compounds. Planting of resistant cultivars seems to be a promising alternative for fruit growers, but there are currently no cultivars available combining resistance to B. cinerea with attractive horticultural traits. Breeding of new cultivars requires the effective identification of resistant strawberry genotypes; therefore the current study was aimed at the evaluation of strawberry genetic resources under controlled conditions by establishing an artificial inoculation assay. The method presented in this study is an artificial inoculation of ripe fruits with a defined spore suspension under laboratory conditions. The results show that this assay is fast and simple and leads to reproducible results that correlate with field observations. Over 3 years a total of 107 strawberry genotypes of the German National Fruit Genebank at the JKI in Dresden‐Pillnitz were evaluated. Five partly resistant genotypes, cultivars Diana, Joerica and Kimberly, and Fragaria virginiana ‘Wildmare Creek’ and F. vesca subsp. bracteata, were identified with mean disease levels of <20% at 6 days post‐inoculation. The obtained results are discussed with regard to future breeding activities.     

哈茨木霉对接种尖孢镰刀茵后黄瓜根系次生代谢物的影响

采用室内筛选与田间试验相结合的方法,对哈茨木霉抑制黄瓜枯萎病菌的拮抗机制进行了研究.对峙培养结果显示木霉菌和病原菌之间形成了较明显的抑菌圈,其中菌株TN对枯萎病菌抑制作用较强.接种木霉菌植株显著提高了植株中肉桂醇脱氢酶（CAD）、多酚氧化酶（PPO）、愈创木酚过氧化物酶（G-POD）、咖啡酸过氧化物酶（CA-POD）和绿原酸过氧化物酶（CGA-POD）的活性及总酚、类黄酮、木质素的含量.接种木霉菌可诱导根系次生代谢相关基因的上调表达,C4H、CAD、CCOMT、G6PDH、PAL和PR-1的表达量分别为对照的 5.64、5.31、4.28、15.33、7.36 和 6.45 倍.表明木霉菌诱导的黄瓜根部对枯萎病的抗性与植物次生代谢密切相关.     

Structure and Expression In planta of Botrytis cinerea Ubiquitin Genes

To identify genes of the necrotrophic pathogenic fungus Botrytis cinerea that are expressed during infection of tomato leaves, a differential screening of a genomic library with radioactively labelled cDNA was performed. This resulted in the identification of a B. cinerea gene, denominated Bcubi4, which encodes a precursor protein consisting of four identical head-to-tail repeats of a 76aa ubiquitin unit. Subsequently a gene denominated Bcubi1CEP79, encoding a single ubiquitin unit joined to a Carboxyl Extension Protein of 79 amino acids, was isolated. The expression of the two ubiquitin genes was studied during pathogenesis of B. cinerea on tomato. Bcubi1CEP79, but not Bcubi4, mRNA was transiently induced at 16h after inoculation. The increased expression of the Bcubi1CEP79 gene at this stage of pathogenesis might be required for enhanced ribosomal biogenesis.     

Evaluation of anilinopyrimidine and other fungicides for control of grey mould (Botrytis cinerea) in container-grown Calluna vulgaris

BACKGROUND:Grey mould (Botrytis cinerea Pers. ex Fr.) is a damaging disease affecting container-grown Calluna vulgaris (L.) Hull. Trials were conducted to test anilinopyrimidine fungicides (pyrimethanil, cyprodinil and mepanipyrim), azoxystrobin and tolylfluanid for crop safety on cultivars, and to evaluate spray programmes based on alternating these fungicides with different modes of action for disease control. Timing and application at different spray intervals were also investigated. RESULTS:Cyprodinil was slightly phytotoxic on only one of the seven cvs tested. Alternating nine-spray programmes of azoxystrobin with an anilinopyrimidine or tolylfluanid, or an anilinopyrimidine with tolylfluanid, with the first spray applied immediately after potting, were equally effective in reducing disease incidence and severity compared with untreated plants. Spray programmes of five or eight sprays, using an anilinopyrimidine and at least one other fungicide with a different mode of action, applied alternately or in blocks of two sprays of the same fungicide and immediately after potting, were equally effective in reducing disease. Omission of early fungicide sprays until 9 weeks after potting and subsequent alternate application of pyrimethanil and tolylfluanid with a different mode of action at 6 week intervals gave reduced disease control. CONCLUSION:There is potential to use spray programmes based on alternating anilinopyrimidines with fungicides with a different mode of action to provide effective control of B. cinerea on C. vulgaris.     

Biosuppression of Botrytis cinerea in grapes

There is increasing interest in the use of biological control agents (BCAs) and plant resistance stimulants to suppress botrytis bunch rot in grapes, caused by Botrytis cinerea . Numerous different filamentous fungi, bacteria and yeasts have been selected as potential BCAs for control of grey mould based upon demonstrated antagonism towards B. cinerea. Biological suppression of the pathogen arises via competition for nutrients and space, the production of inhibitory metabolites and/or parasitism. Preformed and inducible grapevine defence mechanisms also contribute to disease suppression by preventing or delaying pathogenic infection. Furthermore, various biotic and abiotic agents can stimulate grapevine defence mechanisms and so elevate resistance to B. cinerea infection. Biosuppression of B. cinerea in vineyards, using BCAs and resistance stimulants, has been inconsistent when compared with that observed in controlled glasshouse or laboratory conditions. This may be attributable, in part, to the innate variability of the field environment. Research to improve field efficacy has focused on formulation improvement, the use of BCA mixtures and combinational approaches involving BCAs and plant resistance stimulants with complementary modes of action.     

4种杀菌剂及其复配剂对番茄灰霉病菌的毒力

采用菌丝生长速率法测定了咯菌腈、氟啶胺、啶酰菌胺和苯醚甲环唑4种杀菌剂及其两元复配剂对番茄灰霉病菌的毒力。结果显示,咯菌腈、氟啶胺、啶酰菌胺和苯醚甲环唑对番茄灰霉病菌的有效抑制中浓度(EC50)分别为:0.018 0、0.018 1、1.896 8和2.087 4μg/mL。复配剂啶酰菌胺∶苯醚甲环唑1∶5、1∶3和1∶1,咯菌腈∶苯醚甲环唑1∶5增效作用最明显;复配剂咯菌腈∶苯醚甲环唑1∶3,咯菌腈∶氟啶胺1∶3,啶酰菌胺∶咯菌腈5∶1,啶酰菌胺∶苯醚甲环唑3∶1具有增效作用,SR值范围为1.5~4.05,其中以复配剂啶酰菌胺∶苯醚甲环唑1∶5增效作用最好,其SR值为4.05;其余不同配比的各组合复配剂具有相加作用,其SR值范围为0.5~1.46。表明咯菌腈、氟啶胺、啶酰菌胺和苯醚甲环唑4种不同作用机制的杀菌剂可以交替或复配使用,以阻止或延缓灰霉病菌抗药性的进一步加剧,为灰霉病的综合防控和抗药性治理提供理论依据。     

山西省蔬菜灰霉病菌对嘧霉胺的抗药性检测

为明确山西省蔬菜灰霉病菌对嘧霉胺的抗药性,2010-2011年用单孢分离法从山西省数十个县、市蔬菜主产区采集、分离到171株灰霉病病原菌(Botrytis cinerea),并采用最低浓度抑制法测定各菌株对嘧霉胺的敏感性。结果表明,山西省灰霉病菌对嘧霉胺的平均抗性频率为50.29%,平均抗性倍数为1 423.4。在山西省不同地区灰霉病菌对嘧霉胺的抗性存在差异,其中嘧霉胺使用较多的朔州地区抗药性频率达到100%。     

The Role of Trichoderma harzianum Protease in the Biocontrol of Botrytis cinerea

The role of protease of Trichoderma harzianum in the biocontrol of Botrytis cinerea was examined. Two isolates of T. harzianum were compared for their ability to produce protease in liquid culture medium and on the surface of bean leaves. The biocontrol agent T. harziaum T39 produced 58mU/ml of protease and T. harzianum NCIM1185 produced 54mU/ml on the 5th day of growth in liquid culture medium. On bean leaves, combinations of B. cinerea and T. harzianum isolates were examined for the synthesis of protease. The protease activities were 0.9 and 0.6mU/ml for T. harzianum T39 and NCIM1185, respectively, and 0.5mU/ml for B. cinerea alone after 48h of incubation. In the presence of T. harzianum T39 culture liquid containing protease, a 55% reduction in B. cinerea germination and a 80% reduction in the germ tube length were observed after 17h of incubation in vitro. When T. harzianum isolates were added to B. cinerea on bean leaves, increased synthesis of protease was observed (1.0 and 1.2mU/ml for T39 and NCIM1185, respectively). In the presence of T. harzianum NCIM1185 protease, although the rate of germination was reduced, B. cinerea attained 98% germination after 17h of incubation. The hydrolytic enzymes produced by B. cinerea, endo-polygalacturonase (PG) and exoPG were partially deactivated by protease from the T. harzianum isolates. Carboxymethyl cellulase was deactivated only by protease of NCIM1185. On the surface of bean leaves, the protease (obtained from liquid culture medium of T. harzianum isolates) resulted in 56–100% reduction of disease severity. The culture liquid containing protease synthesized on the surface of bean leaves treated with B. cinerea and with T. harzianum was collected and added to fresh leaves infected by B. cinerea. There was 56–100% and 30–75% reduction of disease severity with liquid droplet collected from the leaves treated with T. harzianum T39 and NCIM1185, respectively. Increased control of disease was obtained by combining the conidia of T. harzianum isolates with protease obtained from culture media. Protease inhibitors, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64), antipain hydrochloride, and a mixture of inhibitors, but not pepstatin A, fully or partially nullified the biocontrol effect of T39. T39 was found to be a poor producer of chitinase and -1,3-glucanase in vitro. These enzymes were not detected on leaves treated with T39. Involvement of protease in biocontrol of B. cinerea is suggested.     

Genetic structure and fungicide sensitivity of Botrytis cinerea populations isolated from grapevine in northern Italy

Grey mould, caused by Botrytis cinerea, is a disease severely affecting grape production in northern Italy. However, little information is available on the variability of B. cinerea populations associated with grapevine. The mode of reproduction, sensitivity to fungicides, and for the first time in Italy, the genetic structure of B. cinerea populations isolated from grapevine in a northern Italian region are reported. Botrytis cinerea isolates (317) were completely genotyped for six microsatellite loci and characterized for the presence of the transposable elements Boty and Flipper, for the mating type and for resistance to cyprodinil, fludioxonil, boscalid and fenhexamid. All the isolates were found to belong to B. cinerea Group II, indicating the absence of B. pseudocinerea in the investigated areas. The populations possess a high genotypic diversity, different frequencies of transposable elements and a mixed mode of reproduction. At a regional level, B. cinerea populations belong to a large and interconnected pathogen population that includes the major grape‐growing districts. The populations were generally sensitive to fungicides, with a low proportion (8%) of isolates resistant to cyprodinil, fludioxonil and boscalid. A small genetic distance was found between B. cinerea populations. However, the populations geographically isolated from the others by a mountain range showed a small but statistically significant genetic differentiation and a different pattern of fungicide resistance. The results show that northern Italian B. cinerea populations possess a high evolutionary potential and adaptive capacity.     

河南省番茄灰霉病菌对3种杀菌剂的抗药性检测

为了明确河南省番茄灰霉病菌对苯并咪唑类杀菌剂多菌灵、二甲酰亚胺类杀菌剂腐霉利和氨基甲酸酯类杀菌剂乙霉威的抗药性状况,2013年从河南省不同保护地中采集番茄灰霉病病果或病叶,经单孢分离共获得番茄灰霉病菌菌株139株。采用最低抑制浓度法（MIC）测定了其对多菌灵、腐霉利和乙霉威的抗药性。结果显示：番茄灰霉病菌对多菌灵、腐霉利和乙霉威产生抗性菌株的频率分别为81. 29%、80.58%和93.53%; 所测菌株对3类杀菌剂的抗性类型有BenRDicSNPCS、BenRDicRNPCS、BenRDicSNPCR、BenSDicRNPCR、BenSDicSNPCR和BenRDicRNPCR 6种,所占比例分别为2. 88%、3.60%、3.60%、5.04%、13.67%和71. 22%。表明河南省番茄灰霉病菌已对多菌灵、腐霉利和乙霉威产生抗药性,迫切需要筛选新的杀菌剂防治番茄灰霉病。     

Evaluation of Trichoderma harzianum for controlling root rot caused by Phytophthora capsici in pepper plants

The ability of Trichoderma harzianum to control the rotting of pepper ( Capsicum annuum ) plant roots caused by Phytophthora capsici was studied. Interactions between the fungi were assessed in vitro on three culture media (V8c, Czapek and 2% water agar) and in vivo in plants grown in a substrate inoculated with P. capsici and T. harzianum . Studies on mutual antagonism in vitro showed that P. capsici was inhibited by T. harzianum ; however, the intensity of inhibition differed according to the medium used, being greatest on Czapek. Analysis of the fungal populations in the plant growth substrate showed that T. harzianum consistently reduced that of P. capsici over time. This reduction in the pathogen population was associated with a reduction in root rot of between 24 and 76%, although plant growth (dry weight) was still reduced by 21.2–24.7%, compared with the uninoculated control. In the absence of T. harzianum with the same pathogen inoculum levels, the reduction in dry weight was 59.8–68.6%, suggesting that T. harzianum reduced the damage.     

Characterization and genetic analysis of field isolates ofBotryotinia fuckeliana (Botrytis cinerea) resistant to dichlofluanid

Field isolates ofBotryotinia fuckeliana were collected from naturally infected plants. Their responses to the multisite fungicide dichlofluanid in mycelium growth test fell into three phenotypic classes, characterized by the following EC₅₀ (and MIC) values ing ml^–1: sensitivity, 1–3 (6–10); low resistance, 3–10 (> 100); high resistance, 10–30 (> 100). The corresponding values obtained for these classes in a spore germination test were respectively: 0.05 (0.2), 0.05–0.1 (0.5), 0.5–1 (0.9–1.5). Resistant isolates were crossed with two sensitive and two resistant strains of appropriate mating type to determine the genetic basis of resistance. Distribution of resistance phenotypes in ascospore progeny indicated that a gene, namedDic1, was probably responsible for the low or high resistance of 14 mutants selectively collected from experimental plots of greenhouse-grown gerbera sprayed several times with dichlofluanid or tolyfluanid. A second gene, namedDic2, was probably responsible for the low resistance displayed by two isolates (from grapevine and from carnation) maintained in the laboratory collection. As a result of the investigation, the use of dichlofluanid in integrated management programmes against grey mould is discussed.     

Resistance profiles of Botrytis cinerea populations to several fungicide classes on greenhouse tomato and strawberry in Lebanon

Tomato and strawberry are the most important protected crops in Lebanon and are seriously affected by grey mould disease, caused by Botrytis cinerea. In the present study, the fungicide sensitivity assays revealed medium to high frequencies of B. cinerea isolates resistant to benzimidazoles, dicarboximides, and anilinopyrimidines on tomato and strawberry. Fludioxonil- and boscalid-resistant mutants were uncommonly found at generally low frequency on both crops. Resistance to fenhexamid was detected in only one site on tomato but in most sites on strawberry with high frequencies, and the occurrence of resistance to QoI fungicides was ascertained on both crops. The majority of the tested isolates (>90%) exhibited multiple fungicide resistance, and isolates resistant to the seven antibotrydial fungicide classes were detected on strawberry in three locations. A high level of resistance was shown by B. cinerea mutants resistant to boscalid, fenhexamid, and QoI fungicides, while two levels of moderate and high resistance to anilinopyrimidines were identified. Genetic analysis revealed point mutations in the target genes commonly associated with resistance in B. cinerea isolates, with all mutants resistant to dicarboximides, fenhexamid, boscalid, and QoI fungicides carrying single-nucleotide polymorphims in BcOS1 (I365S/N, Q369P, and N373S), Erg27 (F412V/I), SdhB (H272R/Y), and cytb (G143A) genes, respectively. The general incorrect use of fungicides has caused the development and spread of fungicide resistance as a widespread phenomenon on protected tomato and strawberry in Lebanon. The implementation of appropriate antiresistance strategies is highly recommended.     

Induced systemic resistance in Trichoderma harzianum T39 biocontrol of Botrytis cinerea

Biocontrol of Botrytis cinerea with Trichoderma spp. is generally believed to result from direct interaction of the biocontrol agent with the pathogen or from a Trichoderma-induced change in environmental conditions that affects B. cinerea development. In this work we provide arguments for the participation of induced plant defence in T. harzianum T39 control of B. cinerea. In tomato, lettuce, pepper, bean and tobacco, T. harzianum T39 application at sites spatially separated from the B. cinerea inoculation resulted in a 25–100%percnt; reduction of grey mould symptoms, caused by a delay or suppression of spreading lesion formation. Given the spatial separation of both micro-organisms, this effect was attributed to the induction of systemic resistance by T. harzianum T39. The observation that in bean the effect of T. harzianum T39 was similar to that of the rhizobacterium Pseudomonas aeruginosa KMPCH, a reference strain for the induction of systemic resistance, confirmed this hypothesis. Since B. cinerea control on tobacco leaves sprayed with T. harzianum T39 was similar to the control on leaves from T. harzianum T39 soil-treated plants, induction of plant defence might also participate in biocontrol on treated leaves.     

Evaluation of the incidence of the G143A mutation and cytb intron presence in the cytochrome bc-1 gene conferring QoI resistance in Botrytis cinerea populations from several hosts

BACKGROUND: Previous studies have shown that resistance of Botrytis cinerea to QoI fungicides has been attributed to the G143A mutation in the cytochrome b (cytb) gene, while, in a part of the fungal population, an intron has been detected at codon 143 of the gene, preventing QoI resistance. During 2005–2009, 304 grey mould isolates were collected from strawberry, tomato, grape, kiwifruit, cucumber and apple in Greece and screened for resistance to pyraclostrobin and for the presence of the cytb intron, using a novel real‐time TaqMan PCR assay developed in the present study. RESULTS: QoI‐resistant phenotypes existed only within the population collected from strawberries. All resistant isolates possessed the G143A mutation. Differences were observed in the genotypic structure of cytb. Individuals possessing the intron were found at high incidence in apple fruit and greenhouse‐grown tomato and cucumber populations, whereas in the strawberry population the intron frequency was lower. Cultivation of QoI‐resistant and QoI‐sensitive isolates for ten culture cycles on artificial nutrient medium in the presence or absence of fungicide selection showed that QoI resistance was stable. CONCLUSIONS: The results of the study suggest that a high risk for selection of QoI‐resistant strains exists in crops heavily treated with QoIs, in spite of the widespread occurrence of the cytb intron in B. cinerea populations. The developed real‐time TaqMan PCR constitutes a powerful tool to streamline detection of the mutation by reducing pre‐ and post‐amplification manipulations, and can be used for rapid screening and quantification of QoI resistance. Copyright © 2011 Society of Chemical Industry     

Changes in susceptibility of bean leaves (Phaseolus vulgaris) to Sclerotinia sclerotiorum and Botrytis cinerea by pre-inoculative ozone exposures

The effects of ozone on the susceptibility of leaves ofPhaseolus vulgaris toSclerotinia sclerotiorum andBotrytis cinerea have been investigated. Seedlings of one ozone-sensitive (Pros) and five relatively ozone-insensitive cultivars (Gamin, Precores, Groffy, Narda, Berna) were exposed to different ozone concentrations (0, 120, 180 and 270 g m^–3) for 8 h. One day after the exposures, primary leaves were detached and immediately inoculated with spores of either pathogen suspended in water or in a 62.5 mM KH₂PO₄ (Pi) solution. Visible ozone injury differed between the cultivars and increased with increasing ozone concentration. On the leaves of non-exposed plants, spores of the pathogens suspended in water caused very few lesions, whereas fungal pathogenicity was stimulated by addition of Pi to the inoculum. Ozone-injured leaves of all cultivars exhibited lesions after inoculation of the leaves with the pathogens suspended in water, and the number of lesions was positively correlated with the level of ozone injury for either pathogen and cultivar. The increase in susceptibility of bean leaves in response to increasing ozone concentrations was greater forB. cinerea than forS. sclerotiorum when spores were suspended in water, but was similar when the spores were suspended in Pi.In general, the number of lesions following inoculation with spores in Pi increased with increasing ozone concentration. However, the number of lesions in the ozone-insensitive Groffy was reduced by an exposure to 120 g m^–3 but increased with higher concentrations. This pattern of susceptibility response to the pathogens was not found in the other ozone-insensitive cultivars and, thus, did not appear to be related to the inherent ozone-insensitivity in bean.     

Spatial and temporal variation of reactive oxygen species and antioxidant enzymes in o-hydroxyethylorutin-treated tomato leaves inoculated with Botrytis cinerea

Pretreatment of detached tomato leaves with o -hydroxyethylorutin reduced the percentage leaf area affected, and delayed the appearance of necrosis, following inoculation with conidial suspensions in droplets of the grey mould fungus Botrytis cinerea . o -Hydroxyethylorutin delayed, but did not inhibit, in vitro germination of conidia, although overall percentage germination was reduced compared with water controls. Both the reactive oxygen species (ROS) – superoxide anions and hydrogen peroxide – increased twice as much in o -hydroxyethylorutin-treated leaf tissue 2 and 6 h postinoculation with B. cinerea conidia in tissues under inoculation drops, as well as in surrounding tissues, whereas in plants not pretreated with the compound ROS generation was noticed later, and only in tissues under inoculation drops. Compared with these compounds, changes in the levels of hydroxyl radicals, lipid peroxidation and the activity of the enzymes superoxide dismutase, guaiacol peroxidase, ascorbate peroxidase and catalase were largely unchanged. In stimulating ROS in inoculated tomato tissue, B. cinerea appeared to be affected directly pre- and postinfection, but indirect effects increasing host resistance cannot be excluded.