Peroxidase isoenzymes in the defense response of Capsicum annuum to Phytophthora capsici

Quantitative and qualitative changes in isoperoxidase patterns from stems of three cultivars of pepper ( Capsicum annuum L.). one susceptible, one intermediate and one resistant, were found upon inoculation with Phytophthora capsici using a decapitation method. The peroxidase activity was determined in the intercellular fluid as well as in the cytosolic fraction of the necrotic, healthy and intermediate zones of stems of the three cultivars, 6 days after inoculation. In the intercellular fluid, peroxidase activity of the susceptible cv. Yolo Wonder increased somewhat from 4.7 (healthy zone) to 12.9 (intermediate zone) μmol mg⁻¹ protein min⁻¹, whereas in the intermediate cv. Americano, the peroxidase activity decreased from 123 (healthy zone) to 78 (intermediate zone) μmol mg⁻¹ protein min⁻¹. The most dramatic increase (5.7 to 662 μmol mg⁻¹ protein min⁻¹) in intercellular peroxidase activity was found in the resistant cv. Smith-5. This, in conjunction with the appearance of an additional acidic isoperoxidase (pI 4.4) specific for the cv. Smith-5, could be the reason for the resistance of this cultivar against the fungus attack. The release of peroxidase into the intercellular space as a defense reaction was confirmed by histochemical analysis, showing that peroxidase activity occurred in the intercellular spaces of those stems of the resistant cultivar that had not yet been invaded by the fungus, but was detected neither in the other cultivars nor in the intercellular spaces of such stems of the intermediate and susceptible cultivars that contained growing mycelium of P. capsici. The lack of staining in the intercellular spaces of the susceptible cultivars could be attributed to their low content in peroxidase.     

Defence response of pepper (Capsicum annuum) suspension cells to Phytophthora capsici

Cell suspension cultures of three cultivars of Capsicum annuum L., with different degrees of sensibility to the fungus Phytophthora capsici, responded to elicitation by both lyophilized mycelium and fungus filtrate. They showed conductivity changes, browning, production of the phytoalexin capsidiol and synthesis or accumulation of pathogenesis-related (PR) proteins with glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) activities. The cultivation medium was optimised for growth of both the plant and the fungus in order to avoid any stress during their combination. The resistant cv. Smith-5, showed a more rapid and intense response to the elicitor preparations than the sensitive cvs Americano and Yolo Wonder. This was particularly evident when the cell suspensions were elicited with the filtrate, when differences became clearly visible after only 6 h incubation. The greatest rate of capsidiol accumulation occurred after 18 h in the mycelium-elicited cells and after 12 h in those elicited with the filtrate. These times are the optimal for capsidiol accumulation, and the phytoalexin is produced much more rapidly than it can be excreted into the extracellular medium. The inhibition threshold of fungal growth (300 µg capsidiol [g dry weight]^?1) was reached only in the resistant cultivar. The induction of an intracellular glucanase (pI 8.9 and R_f 0.18) and an extracellular chitinase (pI 5.4 and R_f 0.70) only in the resistant cultivar 24 h after elicitation suggests that these enzymes are involved in the resistance to Phytophthora capsici, while other hydrolases common to all three cultivars form part of a more general defence. The results indicate that elicitation of pepper cell suspension cultures by signal molecules from P. capsici exhibits properties of a multicomponent dynamic system in which different protective mechanisms play complementary roles in the overall expression of the defence reaction. We confirm that the differential responses of resistant and susceptible pepper cultivars to P. capsici previously seen in plant stem sections are retained in suspension culture.     

Induction of sesquiterpenoid biosynthesis in tobacco cell suspension cultures by fungal elicitor

Large amounts of the sesquiterpenoid capsidiol accumulated in the media of tobacco (Nicotiana tabacum L. cv KY14) cell suspension cultures upon addition of fungal elicitor. Capsidiol accumulation was proportional to the amount of elicitor added. The accumulation of capsidiol was preceded by a transient increase in the capsidiol de novo synthesis rate as measured by the incorporation of exogenous [¹⁴C]acetate. Changes in 3-hydroxy-3-methylglutaryl-CoA reductase activity (HMGR; EC 1.1.1.34), an enzyme of general isoprenoid metabolism, paralleled the changes in [¹⁴C]acetate incorporation into capsidiol. Incubation of the cell cultures with mevinolin, a potent in vitro inhibitor of the tobacco HMGR enzyme activity, inhibited the elicitor-induced capsidiol accumulation in a concentration dependent manner. [¹⁴C]Acetate incorporation into capsidiol was likewise inhibited by mevinolin treatment. Unexpectedly, [³H] mevalonate incorporation into capsidiol was also partially inhibited by mevinolin, suggesting that mevinolin may effect secondary sites of sesquiterpenoid biosynthesis in vivo beyond HMGR. The data indicated the importance of the induced HMGR activity for capsidiol production in elicitor-treated tobacco cell suspension cultures.     

Variation in Capsidiol Sensitivity between Phytophthora infestans and Phytophthora capsici Is Consistent with Their Host Range

Plants protect themselves against a variety of invading pathogenic organisms via sophisticated defence mechanisms. These responses include deployment of specialized antimicrobial compounds, such as phytoalexins, that rapidly accumulate at pathogen infection sites. However, the extent to which these compounds contribute to species-level resistance and their spectrum of action remain poorly understood. Capsidiol, a defense related phytoalexin, is produced by several solanaceous plants including pepper and tobacco during microbial attack. Interestingly, capsidiol differentially affects growth and germination of the oomycete pathogens Phytophthora infestans and Phytophthora capsici, although the underlying molecular mechanisms remain unknown. In this study we revisited the differential effect of capsidiol on P. infestans and P. capsici, using highly pure capsidiol preparations obtained from yeast engineered to express the capsidiol biosynthetic pathway. Taking advantage of transgenic Phytophthora strains expressing fluorescent markers, we developed a fluorescence-based method to determine the differential effect of capsidiol on Phytophtora growth. Using these assays, we confirm major differences in capsidiol sensitivity between P. infestans and P. capsici and demonstrate that capsidiol alters the growth behaviour of both Phytophthora species. Finally, we report intraspecific variation within P. infestans isolates towards capsidiol tolerance pointing to an arms race between the plant and the pathogens in deployment of defence related phytoalexins.     

Differences in the Induction of Defence Responses in Resistant and Susceptible Muskmelon Plants Infected with Colletotrichum lagenarium

Defence reactions occurring in resistant (cv. Gankezaomi) and susceptible (cv. Ganmibao) muskmelon leaves were investigated after inoculating with Colletotrichum lagenarium. Lesion restriction in resistant cultivars was associated with the accumulation of hydrogen peroxide (H₂O₂). The activity of antioxidants catalase (CAT) and peroxidase (POD) significantly increased in both cultivars after inoculation, while levels of both CAT and POD activity were significantly higher in the resistant cultivar. Ascorbate peroxidase (APX) increased in both cultivars after inoculation, and level of APX activity was significantly higher in the resistant cultivar. Glutathione reductase (GR) activity significantly increased in both cultivars following inoculation, but was higher in the resistant cultivar, resulting in higher levels of ascorbic acid (AsA) and reduced glutathione (GSH). Phenylalanine ammonia lyase (PAL) significantly increased in inoculated leaves of both cultivars, resulting in higher levels of total phenolic compounds and flavonoids. The pathogenesis‐related proteins chitinase (CHT) and β‐1, 3‐glucanase (GLU) significantly increased following inoculation with higher activity in the resistant cultivar. These findings show that resistance of muskmelon plants against C. lagenarium is associated with the rapid accumulation of H₂O₂, resulting in altered cellular redox status, accumulation of pathogenesis‐related proteins, activation of phenylpropanoid pathway to accumulation of phenolic compounds and flavonoids.     

Differential activation of defense-related genes in susceptible and resistant pepper cultivars infected with Phytophthora capsici

This study investigated the expression pattern of genes encoding for a basic PR-1 protein, a basic beta-1,3-glucanase, a peroxidase, and a sesquiterpene cyclase involved in defense responses in three pepper cultivars with different levels of resistance to Phytophthora capsici. All genes were up-regulated in infected stems of the pepper cultivars, with expression being detected 8h post-inoculation. mRNA levels of these genes increased markedly by 24h post-inoculation, and maximal induction levels were observed for the PR-1 and sesquiterpene cyclase genes. PR-1, peroxidase, and sesquiterpene genes were always expressed at higher levels in resistant cultivars than in the susceptible cultivar, although up-regulation was observed in both, suggesting that the differences between these pepper genotypes in susceptibility and resistance are a matter of the timing and magnitude of the defense response.     

The Effect of Temperature on Symptom Expression and Colonization in Resistant and Susceptible Carnation Cultivars Infected with Fusarium oxysporum f. sp. dianthi

Six commercial carnation cultivars were inoculated with Fusarium oxysporum f. sp. dianthi race 2, and grown under three different temperature regimes. Colonization by the pathogen and development of wilt symptoms were assessed at intervals up to 40 days. No symptoms and very little colonization were seen in any of the cultivars at 14–15°C. At a temperature of 22°C, the cultivars were clearly differentiated into three groups: those with resistance, partial resistance or susceptibility to the pathogen depending on the severity of symptoms and the extent of fungal colonization. Symptom severity was associated with the extent of colonization. This differentiation was not seen at 26°C, when all cultivars except the most resistant, cv.‘Carrier 929′, rapidly became diseased and died by 23 days after inoculation. ‘Carrier 929’ also showed some wilt symptoms at this temperature and was colonized throughout the height of the stem after 40 days. The pathogen caused disease at 26°C by a combination of vascular wilting and stem base and root rotting. Fungal colonization was assayed at 22°C by the dilution plate/homogenization method and by estimation of fungal chitin in a highly resistant (‘Carrier 929′) and in a highly susceptible (‘Red Baron’) cultivar. Both methods of assay gave similar results. In ‘Red Baron’, colonization increased slowly up to 20 days after inoculation then progressed rapidly, closely following the development of severe wilt symptoms. In ‘Carrier 929’, colonization remained very low. The low level of fungal biomass in ‘Carrier 929’ compared with ‘Red Baron’ indicated that the former cultivar showed true resistance as opposed to tolerance to the disease.     

Different Effect of Humidity on Growth and Salt Tolerance of Two Soybean Cultivars

Two soybean (Glycine max (L.) Merr.) cultivars, Tachiyutaka and Dare, were grown in pots at 30 and 70 % relative humidity (RH) and treated with 0 (control), 40 (moderate), 80 and 120 (severe) mM NaCl for 3 weeks. Increasing RH enhanced growth of salt sensitive cultivar, Tachiyutaka, but had no effect on salt tolerant cultivar, Dare, under control and moderate saline conditions. Both cultivars benefited from elevated humidity under severe saline conditions. Cultivar Tachiyutaka had poorer ability for controlling translocation of Na⁺ to the leaves, lower Na⁺ exclusion ability in the roots, and lower root activity under NaCl treatment, compared with cv. Dare. The increased growth of cv. Tachiyutaka at high RH was consistent with decreased Na⁺ accumulation in the leaves, increased stomatal conductance and root activity, while the unchanged growth of cv. Dare was consistent with similar Na⁺ accumulation in the leaves, and the decreased root activity.     

Elicitation of capsidiol accumulation in suspended callus cultures of capsicum annuum

Capsidiol was elicited in suspended callus cultures of Capsicum annuum in response to commercial cellulase (ex Trichoderma viride), or pectinase (ex Aspergiltus niger), or a sterile extract from Gliocladium deliquescens. Amounts of capsidiol up to 2.9 mg per 100 ml of culture were accumulated in response to the G. deliquescens extract. Capsidiol was the preponderant phytoalexin produced in the cultures: minor congeners were present at levels below 0.1% of the amounts of capsidiol.     

Comparative proteomic analysis of methyl jasmonate‐induced defense responses in different rice cultivars

Jasmonate is an important endogenous chemical signal that plays a role in modulation of plant defense responses. To understand its mechanisms in regulation of rice resistance against the fungal pathogen Magnaporthe oryzae, comparative phenotype and proteomic analyses were undertaken using two near‐isogenic cultivars with different levels of disease resistance. Methyl‐jasmonate (MeJA) treatment significantly enhanced the resistance against M. oryzae in both cultivars but the treated resistant cultivar maintained a higher level of resistance than the same treated susceptible cultivars. Proteomic analysis revealed 26 and 16 MeJA‐modulated proteins in resistant and susceptible cultivars, respectively, and both cultivars shared a common set of 13 proteins. Cumulatively, a total of 29 unique MeJA‐influenced proteins were identified with many of them known to be associated with plant defense response and ROS accumulation. Consistent with the findings of proteomic analysis, MeJA treatment increased ROS accumulation in both cultivars with the resistant cultivar showing higher levels of ROS production and cell membrane damage than the susceptible cultivar. Taken together, our data add a new insight into the mechanisms of overall MeJA‐induced rice defense response and provide a molecular basis of using MeJA to enhance fungal disease resistance in resistant and susceptible rice cultivars.     

Symbiotic nitrogen fixation and nitrate reduction in two peanut cultivars with different growth habit and branching pattern structures

We have investigated the response of two peanut cultivars (TEGUA and UTRE) with different growth habits and branching pattern structures to different nitrogen (N) sources, namely, N-fertilizer or N₂ made available by symbiotic fixation, and analysed the pattern of nitrate reductase (NR) activity in these cultivars. Nitrate and amino acid contents were also examined under these growth conditions. In terms of nitrogen source, cv. TEGUA showed a better response to inoculation with Bradyrhizobium sp. SEMIA 6144 at 40 days after planting, while cv. UTRE responded better to N-fertilizer (5 mM KNO₃). Both cultivars showed different patterns of NR activity in the analyzed plant organs (leaves, roots, and nodules), which were dependent on the N source. When nitrogen became available to the plant through symbiotic N₂ fixation, the patterns of NR activity distribution were different in the two cultivars, with cv. TEGUA showing a higher NR activity in the nodules than in the leaves and roots, and cv. UTRE showing no difference in terms of NR activity among organs. The nitrate and amino acid contents showed a similar trend between the two cultivars, with the highest nitrate content in the leaves of fertilized plants and the highest amino acid content in the nodules. The high nitrate content of the leaves of cv. UTRE indicated the better response of this cultivar to N-fertilizer.     

A comparison of inoculation methods for determining potato cultivar reaction to black leg

Two methods of inoculating tubers, one by dipping them in an aqueous suspension of Erwinia carotovora subsp. atroseptica, the other by inserting the end of a wooden toothpick charged with undiluted bacteria into the rose-end near a sprout, were compared over two years for their ability to produce black leg and for their effect on plant growth and yield on a number of cultivars chosen because of expected differences in susceptibility. Although both methods produced a similar rank order for cultivar reaction, the more consistent results were obtained using the toothpick method, clear differences in black leg incidence and yield being evident between the susceptible cultivars Ulster Sceptre and Maris Bard and the more resistant Pentland cultivars. As some cultivars appear more tolerant to black leg than others, yield may be as important a criterion as disease incidence when assessing overall cultivar performance. The inoculation of the base and middle of stems and the infiltration of the bacterium into tubers were also investigated as alternative methods for assessing cultivar susceptibility.     

Effects of Salinity on the Extensibility and Ca Availability in the Expanding Region of Growing Barley Leaves

The growth of barley (Hordeum vulgare L.) leaves is reduced by salinity. We used the Instron extensometric technique to measure the reversible and irreversible compliance of the expanding regions of growing barley leaves from plants exposed to 1, 40, 80 and 120 mM NaCl in nutrient solution. Two barley cultivars differing in salinity resistance (cv ‘Arivat’ and cv ‘Briggs’) were compared over 5d of leaf growth. During the period of most active leaf expansion, salinity reduced reversible compliance and increased compliance in the leaf segments, although responses to salinity were complex and changed over the course of leaf expansion. Salinity increased irreversible compliance more in the salt-sensitive cultivar Arivat than in the more salt-tolerant cultivar Briggs. Elemental analysis of the basal leaf segments used for extensometry revealed an accumulation of Na and a depletion of Ca in segments from salinized plants, resulting in very high Na: Ca ratios in salinized expanding tissue. The concentrations of K and Mg in basal leaf tissue were elevated by salinity. Our data do support the hypothesis that the inhibition of leaf expansion by salinity stress is mediated by a decline in irreversible extensibility. We suggest that reduced Ca availability in expanding leaf tissue may contribute to growth reduction in salt-stressed barley seedlings.     

Silicon ameliorates manganese toxicity by regulating manganese transport and antioxidant reactions in rice (Oryza sativa L.)

Background and aims

This study aimed to investigate the roles of silicon (Si) in ameliorating manganese (Mn) toxicity in two rice (Oryza sativa L.) cultivars: i.e. cv. Xinxiangyou 640 (XXY), a Mn-sensitive cultivar and cv. Zhuliangyou 99 (ZLY), a Mn-tolerant cultivar.

Methods

Plants were cultured in nutrient solution containing normal Mn (6.7 μM) or high Mn (2.0 mM), both with or without Si supply at 1.5 mM Si.

Results

Plant growth was severely inhibited by high Mn in cv. XXY, but was enhanced by Si supply. In cv. XXY, Si-enhanced tolerance resulted from a restriction of Mn transport, whereas in cv. ZLY Mn uptake was depressed. In cv. XXY, high Mn significantly increased superoxide dismutase (SOD), catalase and ascorbate peroxidase activities but decreased non-protein thiols and glutathione concentrations, leading to accumulation of H₂O₂ and malondialdehyde. The addition of Si significantly counteracted high Mn-elevated malondialdehyde and H₂O₂ concentrations and enhanced plant growth. In cv. ZLY, high Mn considerably raised SOD activities and glutathione concentrations, thus leading to relatively low oxidative damage.

Conclusions

Si-enhanced Mn tolerance was attributed mainly to restricted Mn transport in cv. XXY but to depressed Mn uptake in cv. ZLY. Silicon mainly influenced non-enzymatic antioxidants in these two rice cultivars under high Mn stress.     

The effect of salt stress on lipid peroxidation, antioxidative enzymes and proline content of sesame cultivars

The effect of increasing NaCl concentrations was studied on two different cultivars (cv. Orhangazi and cv. Cumhuriyet) of Sesamum indicum. Seedlings were grown for 40 days in half strength Hoagland solution and after 40 days treated with different NaCl concentrations (0, 50 and 100 mM) for 21 days. Differences in growth parameters, lipid peroxidation, antioxidative enzyme activities and proline accumulation were tested in order to put forward the relative tolerance or sensitivity of the cultivars. Results indicated that both parameters differ according to the cultivar's ability in coping oxidative stress caused by salinity. Constitutive levels of antioxidative enzyme activities were almost the same between the cultivars; however, cv. Cumhuriyet was able to induce antioxidative enzyme activities more efficiently when subjected to salt stress. Growth parameters, lipid peroxidation and proline accumulation results are also in good correlation with supporting this cultivar's being relatively tolerant.     

Growth stage-based modulation in antioxidant defense system and proline accumulation in two hexaploid wheat (Triticum aestivum L.) cultivars differing in salinity tolerance

Changes in the antioxidant defense system and proline accumulation were examined at different growth stages (vegetative, boot and reproductive) in plants of two hexaploid spring wheat cultivars (S-24, salt tolerant; MH-97, salt sensitive), grown in hydroponics and salinity-affected with 0, 50, 100 and 150 mM of NaCl. Salt stress provoked a marked decline in plant dry mass and ascorbic acid contents, and increased proline, total soluble proteins and H₂O₂ contents in both wheat cultivars at all growth stages. However, higher accumulation of proline and H₂O₂ contents was recorded at the vegetative and boot stages, respectively, in both wheat cultivars. Salt stress caused a consistent rise in the activities of some key antioxidant enzymes (CAT, SOD, POD, and APX) at all growth stages only in the salt tolerant cultivar S-24, whereas such pattern of enhanced activities of enzymatic antioxidants in cv. MH-97 was found only at the vegetative stage under saline regimes. Maximum activities of various enzymatic antioxidants were observed at the vegetative stage in both wheat cultivars under varying external salt treatments. The results showed that high salinity tolerance of cv. S-24, as manifested by lower decrease in its dry matter under salt stress, was associated with higher activities of antioxidant enzymes, increased accumulation of proline, and lower levels of H₂O₂, as compared with cv. MH-97 at all growth stages under saline regimes.     

Introduction a potato cultivar "sprit" as relatively resistant to main fungal pathogens causal agents of early blight and wilting on potato in Iran

Potato (Solanum tubersum L.) is one of the most human food production cultured in Iran especially Zanjan province as a temperate region. Some fungal pathogens caused severely infected on potato tubers or foliage in the majority grown areas and resulted yield losses in potato production. Recent years from 2002 to 2004 infected samples were collected from different potato grown regions in Zanjan province then cultured on PDA after surface sterilization with sodium hypochlorite. Isolated fungal pathogens were identified and study showed the main pathogens with high incidence and frequency were Alternaria solani, Fusarium oxysporum and Verticillium sp. in studied areas. The regions which used convention varieties showed more diseases than other locations which used relatively resistant races. The rate of resistance for 10 international potato varieties was studied by inoculation of them by 10(5) spores suspension of three common fungal pathogens in the field. Study showed Sprit cultivar was more resistant than others to all three common pathogens and Lady-Claire was most susceptible. Yield production of Sprit per unit of land area was also exceeded that of other cultivars by factors of 1.10 to 2.25 respectively. The results of the study helped potato growers to culture Sprit cultivar and have good yield production in Zanjan and Hamedan provinces in this year.     

Phenylalanine Ammonia-lyase Activity in Barley After Infection with Bipolaris sorokiniana or Treatment with its Purified Xylanase

Phenylalanine ammonia-lyase (PAL) activity was determined from leaves and roots of two barley (Hordeum vulgare L.) cultivars after infection with a necrotrophic pathogen, Bipolaris sorokiniana (Sacc.) Shoem., and treatment with its purified xylanase. PAL activity increased in leaves of both cultivars 16 h after fungal inoculation but two phases, with activity peaks at 24–32 h and 40 h, were recorded only for the more resistant cultivar, Agneta. Attempts to use a PAL inhibitor, χ-amin, ooxyacetic acid, to increase susceptibility to B. sorokiniana in barley leaves were unsuccessful. Treatments of leaves with purified xylanase resulted in more rapid (4–12 h after injection), although reduced, induction of PAL compared with fungal injection. The higher the concentration of xylanase applied the earlier the activity peaks were detected. Fungal inoculation only slightly increased PAL activity in barley roots while xylanase treatment had no effect. The basal level of PAL was however much higher in roots than in leaves. In wheat, Triticum aestivum L. resistant to B. sorokiniana, the time-course of PAL induction after fungal infection and xylanase treatment resembled that for cv. Agneta, while in oats, Avena sativa L. (non-host) PAL activity did not change after the treatments. The results suggest that the second phase of PAL induction, associated only with responses of barley cv. Agneta and wheat, is linked with their resistance to B. sorokiniana infection. The possible role of xylanase as an elicitor of PAL is discussed.