Changes in Sugar Content During Induction of Systemic Acquired Resistance to Late Blight Caused by Phytophthora infestans (Mont.) de Bary in Potato

Resistance of plants to their pathogens may be systemically enhanced by a localized induction treatment, a characteristic named systemic acquired resistance (SAR). Changes in amounts and proportions of sucrose, glucose and fructose were examined in induced and noninduced potato plants ( Solarium luberosum L. ev. Matilda) before, and 1–9 days after challenge inoculation. The potato plants were induced and challenged with Phytophthora infesians (Mont.) de Bary. Plants which were non-induced and non-challenged were also tested (referred to as non-treated). The sugars were analysed using high-performance anion exchange chromatography with pulsed electrochemical detection, HPAE-PED (Dionex system). The amounts as well as the proportions of sucrose, glucose and fructose were similar throughout the experimental period in the non-treated plants. In induced plants, before challenge inoculation, the amount of sucrose was 44% lower than in non-treated plants. One day after challenge inoculation, the amount of sucrose had decreased by 79% in non-induced plants compared with non-treated plants, while the induced plants were much less affected. The total amount of all sugars were approximately the same 1 day hefore and 9 days after the challenge inoculation in ail treatments. However, 1 day after challenge inoculation, a 65% decrease in total amount was observed in the non-induced plants compared with the smaller reduction of 37% in the induced plants. The large differences in sugar amounts and proportions between the different treatments are discussed.     

番茄苗龄对晚疫病生理小种T1的抗性表现差异研究

对感病和抗病品系3、6、9叶苗龄番茄幼苗人工接种晚疫病菌T1小种,结果表明,感病品系随着苗龄的增加,对晚疫病的感病程度有所减轻,其中6叶期较3叶期、9叶期较6叶期幼苗的病级指数分别降低了28.8%和3.9%,但差异不显著;9叶期较3叶期降低了31.0%,差异达显著水平。抗病品系的6叶期和9叶期较3叶期幼苗病级指数均降低了50%左右,差异达极显著水平;6叶期和9叶期幼苗抗性基本相同。     

Antimicrobial Activities of the Essential Oils of Various Plants against Tomato Late Blight Disease Agent Phytophthora infestans

The aim of this study was to find an alternative to synthetic fungicides currently used in the control of devastating oomycete pathogen Phytophthora infestans, causal agent of late blight disease of tomato. Antifungal activities of essential oils obtained from aerial parts of aromatic plants such as oregano (Origanum syriacum var. bevanii), thyme (Thymbra spicata subsp. spicata), lavender (Lavandula stoechas subsp. stoechas), rosemary (Rosmarinus officinalis), fennel (Foeniculum vulgare), and laurel (Laurus nobilis), were investigated against P. infestans. Both contact and volatile phase effects of different concentrations of the essential oils used were determined by using two in vitro methods. Chemical compositions of the essential oils were also determined by GC-MS analysis. Major compounds found in essential oils of thyme, oregano, rosemary, lavender, fennel and laurel were carvacrol (37.9%), carvacrol (79.8), borneol (20.4%), camphor (20.2%), anethole (82.8%) and 1,8-cineole (35.5%), respectively. All essential oils were found to inhibit the growth of P. infestans in a dose-dependent manner. Volatile phase effect of oregano and thyme oils at 0.3 μg/ml air was found to completely inhibit the growth of P. infestans. Complete growth inhibition of pathogen by essential oil of fennel, rosemary, lavender and laurel was, however, observed at 0.4–2.0 μg/ml air concentrations. For the determination of the contact phase effects of the tested essential oils, oregano, thyme and fennel oils at 6.4 μg/ml were found to inhibit the growth of P. infestans completely. Essential oils of rosemary, lavender and laurel were inhibitory at relatively higher concentrations (12.8, 25.6, 51.2 μg/ml respectively). Volatile phase effects of essential oils were consistently found to be more effective on fungal growth than contact phase effect. Sporangial production was also inhibited by the essential oil tested. Light and scanning electron microscopic (SEM) observation on pathogen hyphae, exposed to both volatile and contact phase of oil, revealed considerable morphological alterations in hyphae such as cytoplasmic coagulation, vacuolations, hyphal shrivelling and protoplast leakage.     

Systemically Induced Resistance in Potato Cultivars with Different Degree of Resistance to Late Blight caused by Phytophthora infestans (Mont.) de Bary

Systemically induced resistance in potato plants (Solanum tuberosum L.) against late blight Phytophthora infestans was studied macroscopically on three potato cultivars with different degrees of resistance to late blight. The cultivars, were Bintje(2), Elin(3) and Matilda(4). Figures within brackets represent resistance to leaf late blight on a scale from 1 to 5, where 5 denotes the highest level of resistance. In vitro propagated cuttings were used in the experiments, which were, performed under controlled environmental conditions. Disease resistance was induced by a local pre-infection with P. infestans or with the nonpathogen P. cryptogea. Induced resistance was observed on all cultivars. The protection causedby, induced resistance tended to be most intense on cv. Matilda, with 50–60% reduction in lesion formation 4 days after challenge inoculation.     

Infection Potential of Hairy Nightshade (Solanum sarrachoides) by Phytophthora infestans and Late Blight Implications of the Alternate Host

Infection of hairy nightshade ( Solanum sarrachoides Sendt.) by Phytophthora infestans has been reported. However, the epidemiological significance of hairy nightshade to potato late blight is not well known. Disease development and infection rates of P. infestans were quantified on hairy nightshade relative to tomato (cv. Brandywine) and potato (cv. Shepody) hosts to evaluate infection potential at 14, 18, 22 and 26°C and 72, 82, 87, and 92% relative humidity (RH). The susceptibility of hairy nightshade to inoculum levels, weed ontogeny, and sporangia production potential were also investigated. Late blight development varied among hairy nightshade, tomato and potato hosts. Pathogen infection rates ranged from 0.0325 to 0.4674 gompits/day (unit for quantifying infection rates), and were significantly (P < 0.05) greater on potato and tomato than on hairy nightshade. Late blight severity was variably affected by RH. Disease levels on hairy nightshade varied with inoculum load; and ranged from 9 to 26% and 26 to 37% at low (5 × 10³) and high (25 × 10³) sporangia concentrations, respectively. Late blight was recorded irrespective of hairy nightshade ontogeny, and was significantly greater on 8–10 than 4–6-week-old plants. These results indicate that pathogen, environmental and host factors affect late blight development on hairy nightshade.     

Cellular Responses of the Late Blight Pathogen Phytophthora infestans to Cyclic Lipopeptide Surfactants and Their Dependence on G Proteins

Oomycete pathogens cause major yield losses for many crop plants, and their control depends heavily on agrochemicals. Cyclic lipopeptides (CLPs) were recently discovered as a new class of natural compounds with strong activities against oomycetes. The CLP massetolide A (Mass A), produced by Pseudomonas fluorescens, has zoosporicidal activity, induces systemic resistance, and reduces late blight in tomato. To gain further insight into the modes of action of CLPs, the effects of Mass A on pore formation, mycelial growth, sporangium formation, and zoospore behavior were investigated, as was the involvement of G proteins in the sensitivity of Phytophthora infestans to Mass A. The results showed that Mass A induced the formation of transmembrane pores with an estimated size of between 1.2 and 1.8 nm. Dose-response experiments revealed that zoospores were the most sensitive to Mass A, followed by mycelium and cysts. Mass A significantly reduced sporangium formation and caused increased branching and swelling of hyphae. At relatively low concentrations, Mass A induced encystment of zoospores. It had no effect on the chemotactic response of zoospores but did adversely affect zoospore autoaggregation. A loss-of-function transformant of P. infestans lacking the G-protein α subunit was more sensitive to Mass A, whereas a gain-of-function transformant required a higher Mass A concentration to interfere with zoospore aggregation. Results indicate that Mass A disturbs various developmental stages in the life cycle of P. infestans and suggest that the cellular responses of P. infestans to this CLP are, in part, dependent on G-protein signaling.Oomycetes cause devastating diseases of plants and animals. They are fungal look-alikes that grow as mycelium and propagate via spores but evolved independently from fungi (23). Among the plant pathogens are over 80 Phytophthora species, with the late blight pathogen Phytophthora infestans being the most renowned (12, 16). Late blight control relies heavily on fungicides that contain copper-, tin-, phenylamide-, or cyanocetamide-oximes as active ingredients. Public concerns about the adverse effects of these fungicides on food safety and the environment have led to an increased demand for novel control strategies, preferably based on natural products. In recent years, the destructive effects of cyclic lipopeptides (CLPs) on zoospores of oomycete plant pathogens have attracted considerable attention (8, 9, 33). CLPs are produced by a variety of bacterial genera including Bacillus and Pseudomonas (13, 29, 30, 33, 37). They are composed of a fatty acid tail linked to an oligopeptide, which is cyclized by a lactone ring between two amino acids in the peptide chain. Based on the length and composition of the fatty acid as well as the number, type, and configuration (L-D form) of the amino acids in the peptide moiety, their activity may change (29, 30, 33). CLPs can be chemically produced, and via structural or genetic modifications, their physicochemical properties and antimicrobial activities can be altered (1). Pseudomonas fluorescens strain SS101 produces nine cyclic lipopeptide surfactants, with massetolide A (Mass A) being the main cyclic lipopeptide (7, 9). The others are derivatives of Mass A differing in the amino acid compositions of the peptide ring (7).One of the main modes of action of natural and synthetic CLPs is interference with the membrane integrity of the target organism, leading to pore formation and cytolysis (4, 5, 18, 19, 27, 34). For example, the CLPs Mass A and viscosin, produced by P. fluorescens strains SS101 and SBW25, respectively, act on membranes of zoospores of plant-pathogenic oomycetes, including Pythium and Phytophthora species, and this leads to the complete elimination of these propagules within 1 min of exposure (6-9). The destructive effects of Mass A on zoospores may explain, at least in part, the activity of P. fluorescens SS101 against Pythium root rot of flower bulb crops (6, 9) and tomato late blight caused by P. infestans (44). However, CLPs not only act on zoospores but may also inhibit mycelial growth of oomycetes and fungi (29, 33). The observations that several CLPs induce systemic resistance in plants against fungal and oomycete pathogens including P. infestans (31, 44) further emphasize their versatile activities and potential for crop protection. To explore and exploit the use of CLPs as a novel supplementary strategy for disease control, fundamental knowledge on their modes of action and the cellular responses of target oomycete pathogens is required.In this study, we investigated the response of P. infestans to the CLP Mass A produced by P. fluorescens SS101. We examined various growth stages of P. infestans and performed dose-response experiments to determine the effects of Mass A on mycelial growth, sporangium formation, cyst germination, and zoospore behavior, including chemotaxis, autoaggregation, and encystment. We also examined the involvement of the α subunit of the heterotrimeric G protein in the cellular responses of P. infestans to Mass A. The results show differential sensitivities of the various growth stages to Mass A and suggest that G-protein signaling plays a role in mediating the response.     

Thigmomorphogenesis: the Effect of Mechanical Perturbation and Ethrel on Stem Pithiness in Tomato [Lycopersicon esculentum (Mill.) Plants]

The stems of ‘Y’-shaped (double stemmed) tomato(Lycopersicon esculentum Mill.) plants were mechanically perturbed(MP) by stroking for 6 successive days. The treatment reducedelongation of the two stems by 40 per cent. When only one branchof the pair was treated, its length was reduced to the sameextent as the two branches in the previous treatment, whilethe elongation of the untreated branch was increased by 60 percent over that of the control. Withholding irrigation induced stem pithiness due to droughtstress in non-MP-plants. However, in MP-pretreated plants, thenumber of pithy internodes was markedly less and the degreeof severity of the disorder was reduced. Ethrel applicationmimicked the effects of MP on pithiness. In some unknown way,the plants are hardened by MP or Ethrel. Lycopersicon esculentum Mill, tomato, drought stress, thigmomorphogenesis, ethylene, pithiness     

番茄"白化"突变体果实的几个生理生化特性检测

在番茄“白化”突变体果实发育过程中,总叶绿素、叶绿素a、叶绿素b的含量均小于正常植株的果实;果肉细胞内含有白色体;果实在贮藏期间的硬度较大。而果实品质,如糖酸比、维生素C和可溶性固形物的含量与正常果实差异不大。     

A Potential Defense Mechanism of Tomato Against the Late Blight Disease is Suppressed by Germinating Sporangia-derived Substances from Phytophthora infestans

Hypersensitive browning, host cell death and phytoalexins (rishitin) synthesis in both callus cultures and cotyledons have been observed in a susceptible tomato cultivar following treatment with high molecular weight cell wall components from Phytophthora infestans race 1B. The growth of the pathogen was restricted on both elicited differentiated and undifferentiated tomato tissues. Subtance(s) released by germinating sporangia, but not by the mycelium, suppressed the hypersensitive reaction.     

Utilization of Caffeic Acid Phenethyl Ester to Control Alternaria alternata Rot in Tomato (Lycopersicon esculentum Mill.) Fruit

Chemical fungicides that are related with resistant strains develop negative effects on human health and environment. Propolis is a resinous substance collected by bees with positive effects on human health and inhibitory activity against Alternaria alternata. Caffeic acid phenethyl ester (CAPE) is a component of the propolis. The objective of this experiment was to test the effect of CAPE on fungi infecting tomato fruit using as a model the pathosystem A. alternata‐tomato. CAPE was chemically synthesized in our laboratory and analysed with nuclear magnetic resonance spectroscopy. Different concentrations (0, 16, 32, 48, 64, 80, 90 and 100 μm ) of CAPE were tested on A. alternata growing in vitro. For the in vivo experiment, red ripe tomato fruit was inoculated with A. alternata and untreated or treated with 1, 50 and 100 μm of CAPE. After that, the fruit was stored at 25°C for up to 20 days. Colony size (CS) was recorded in vitro. In tomato fruits, the severity of infection (SI), respiration rate (RR), ethylene production (EP), pH, total soluble solids (TSS), weight loss (WL) and titratable acidity (TA) were evaluated during the storage time. CAPE melting point and spectral data probed to be the right molecule. In vitro, 64 and 100 μm of CAPE reduced CS by 30%. In vivo, 50 and 100 μm of CAPE reduced SI higher than the fungicide Captan^® with no effects on RR, EP, WL, pH, TSS and TA. It was concluded that CAPE controls A. alternata infection better than a commercial fungicide without negative effects on tomato fruit ripening and fruit quality.     

Sunflower (Helianthus annuus L.) Pathogenesis-Related Proteins (Induction by Aspirin (Acetylsalicylic Acid) and Characterization)

Sunflower leaf discs floated on a solution containing aspirin (acetylsalicylic acid) produced a set of new proteins extractable at pH 5.2 and excreted into the intercellular space. More than 80% of the proteins found in the intercellular fluids of induced leaf discs have been identified as pathogenesis-related (PR) proteins by their immunological relationship with tobacco PR proteins. Members of the four major classes of PR proteins have been characterized. Sunflower PR proteins of type 1 (PR1) and of type 3 (PR3) were found to have acidic isoelectric points, whereas the induced PR protein of type 2 (PR2) had a basic isoelectric point. Members of the type 5 PR proteins (PR5), known in tobacco as thaumatin-like proteins, showed a more complex pattern. Multiple sunflower PR5 isomers of similar molecular weight but of different isoelectric points were excreted from the cells in response to the aspirin treatment. PR2 and PR3 proteins were found at very low basal levels in untreated leaves, whereas PR1 and PR5 proteins could not be detected at all in the same extracts. Glucanase and chitinase activities were always associated with PR2 and PR3 proteins in partially purified sunflower extracts. All of these data indicate that, in response to aspirin treatment, sunflower plants produce a complete set of PR proteins characterized by an apparently exclusively extracellular localization.     

喷施己酸二乙氨基乙醇酯对几种与番茄幼苗抗冷性相关生理指标的影响

番茄幼苗叶面喷施适宜浓度(20～40mg·L~(-1))的己酸二乙氨基乙醇酯(DA-6)可有效提高冷胁迫下番茄幼苗植株的抗寒能力。冷胁迫下,20～40mg·L~(-1)DA-6处理的番茄植株自由水/束缚水比值降幅较大,叶中叶绿素含量、超氧化物歧化酶(SOD)、过氧化物酶(POD)和抗坏血酸过氧化物酶(APX)活性也明显增大,丙二醛(MDA)积累下降25.8%～34.6%,可溶性糖和可溶性蛋白质含量提高。     

Characterization of the Stimulation of Ethylene Production by Galactose in Tomato (Lycopersicon esculentum Mill.) Fruit

We have characterized the stimulation of ethylene production by galactose in tomatoes (Lycopersicon esculentum Mill.). The effect of concentration was studied by infiltrating 0, 4, 40, 100, 200, 400, or 800 micrograms galactose for each gram of fresh fruit weight into mature green `Rutgers' fruit. Both 400 and 800 micrograms per gram fresh weight consistently stimulated a transient increase in ethylene approximately 25 hours after infiltration; the lower concentrations did not. Carbon dioxide evolution of fruit infiltrated with 400 to 800 micrograms per gram fresh weight was greater than that of lower concentrations. The ripening mutants, rin and nor, also showed the transient increase in ethylene and elevated CO₂ evolution by 400 micrograms per gram fresh weight galactose. 1-Aminocyclopropane-1-carboxylic acid (ACC) content and ACC-synthase activity increased concurrently with ethylene production. However, galactose did not stimulate ACC-synthase activity in vitro. The infiltrated galactose in pericarp tissue was rapidly metabolized, decreasing to endogenous levels within 50 hours. Infiltrated galacturonic acid, dulcitol, and mannose stimulated transient increases in ethylene production similar to that of galactose. The following sugars produced no response: sucrose, fructose, glucose, rhamnose, arabinose, xylose, raffinose, lactose, and sorbitol.     

Tomato (Lycopersicon esculentum L.) nutrient and lead uptake affected by zeolite and DTPA in a lead‐polluted soil

• Development of alleviation strategies, which enhance plant growth under heavy metal stress, is important. Inorganic (zeolite) and organic (diethylene triamine penta‐acetic acid, DTPA) amendments affecting the alleviation of lead (Pb) stress in a calcareous soil were tested by investigating leaf nutrient uptake of tomato (Lycopersicon esculentum L.) plants.
• Experimental quantities of lead (Pb) at 0, 50, 100 and 150 mg·kg^?1 soil, zeolite (clinoptilolite) at 0%, 0.5% and 1%, and DTPA at 0, 50 and 100 mg·kg^?1 soil were tested in a factorial experiment with three plant replicates.
• According to the anova , Pb, zeolite, DTPA and their interactions significantly affected plant concentrations of nitrogen (N), potassium (K), iron (Fe), zinc (Zn), copper (Cu), manganese (Mn) and lead (Pb). With increasing DTPA concentration at different levels of zeolite and Pb, plant concentrations of macro‐ and micronutrients significantly increased. Increasing soil Pb increased leaf Pb concentration and decreased the uptake of N, K, Fe, Zn, Cu and Mn. Although with increasing Pb concentration the uptake of macro‐ and micronutrients decreased in tomato, the use of zeolite and DTPA alleviated this stress by increasing nutrient uptake compared to the control. Interestingly, however, increased levels of zeolite and DTPA led to a decreased uptake of nutrients by plants (compared with control), indicating the absorption of such nutrients by the two amendments and their partial release for further plant use.
• Zeolite and DTPA may alleviate the negative effects of soil Pb on tomato growth by decreasing nutrient leaching and increasing plant nutrient uptake.

     

An Agar-diffusion Technique Applied to the Study of Gibberellins in Tomato (Lycopersicon esculentumMill.) Plants

Chromatography of extracts of agar blocks on which shoot tipsof tomato had stood for 24 h revealed the presence of at leasttwo gibberellin-like substances. Gibberellin-like activity detectedby the dwarf pea bioassay was shown to be associated with activityin promoting elongation growth in tomato seedlings. The activesubstances were acidic; tests for the presence of ‘neutral’,‘basic’ and ‘bound’ gibberellins inextracts of the agar blocks gave negative results. Amounts ofgibberellin-like substances obtained from the shoot tips wereincreased by the incorporation of EDTA into the agar blocksbut were apparently unaffected by the temperature (within therange 5–25 °C) at which the diffusion was carriedout. It is suggested that the technique measured levels of diffusiblegibberellins in the tissues at the time of their excision ratherthan rates of gibberellin synthesis during the diffusion period.     

Quantification of Indole-3-Acetic Acid in Dark-Grown Seedlings of the Diageotropica and Epinastic Mutants of Tomato (Lycopersicon esculentum Mill.)

Endogenous indoleacetic acid (IAA) levels were examined in 7-day-old, dark-grown tomato seedlings (Lycopersicon esculentum Mill. cv VFN8), and in two single-gene mutants, Epinastic and diageotropica. Gas chromatography-mass spectrometry was employed to quantify IAA using ¹³C₆-[benzene ring]indoleacetic acid as internal standard. IAA concentrations ranged from 89 to 134 nanograms per gram dry weight and were not significantly different for the three genotypes. Ethylene over-production by dark-grown Epi seedlings is not likely to result from increased IAA. Assuming similar recovery percentages for each genotype, indole-3-ethanol, a purported storage form of IAA, was identified by GC-MS and found to be more prevalent in the parent tomato, VFN8, with only trace amounts observed in Epi. No IEt was detected by high performance liquid chromatography/fluorescence in dgt (detection limit >100 picograms).     

Studies on the Changes in the Major Nucleic Acid Components of Tomato Seeds (Lycopersicon esculentum Mill.) Resulting from Osmotic Presowing Treatment

Extensive accumulation of nucleic acid may be one of the factorsinvolved in the acceleration of tomato seed germination by osmoticpresowing treatment. The experiments reported here were designedto characterize the changes in the major nucleic acids duringosmotic pretreatment and subsequent germination. Polyacrylamidegel electrophoresis showed that ribosomal RNA comprised themajor part of this net synthesis, but it was more difficultto ascertain whether or not there was a proportional increasein sRNA, as part of the latter peak may comprise comigratingfragments of degraded rRNA. The mass ratio of 25S: 18S rRNAsextracted from dry seeds was near unity, while during normalgermination and during pretreatment this ratio increased toabout 1?9:I, close to the theoretical ratio assuming 25S and18S rRNAs occur in equimolar proportions. This leads to thespeculation that replacement of old ribosomes may occur duringearly germination and pretreatment, although this would notaccount for all the synthetic activity observed during the latterprocess. Use of the diphenylamine colour reaction showed that no increasein DNA results from pretreatment and that the onset of the firstS-phase during subsequent germination of pretreated seeds wascommensurate with the advancement of the time to 50% germination.