Defence‐Related Enzymes in Soybean Resistance to Target Spot

Target spot, caused by the fungus Corynespora cassiicola, has become a serious foliar disease in soybean production in the Brazilian Cerrado. Information in the literature regarding the biochemical defence responses of soybean to C. cassiicola infection is rare. Therefore, the objective of this study was to determine the biochemical features associated with soybean resistance to target spot. The activities of chitinases (CHI), β‐1‐3‐glucanases (GLU), phenylalanine ammonia‐lyases (PAL), peroxidases (POX), polyphenol oxidases (PPO) and lipoxygenases (LOX), as well as the concentrations of total soluble phenolics (TSP) and lignin‐thioglycolic acid (LTGA) derivatives, were determined in soybean leaves from both a resistant (FUNDACEP 59) and a susceptible (TMG 132) cultivar. The target spot severity, number of lesions per cm² of leaflet and area under the disease progress curve were significantly lower for plants from cv. FUNDACEP 59 compared to plants from cv. TMG 132. The GLU, CHI, PAL, POX and PPO activities and the concentration of LTGA derivatives increased significantly, whereas LOX activity decreased significantly on the leaves infected by C. cassiicola. Inoculated plants from cv. FUNDACEP 59 showed a higher PPO activity and concentrations of TSP and LTGA derivatives at 4 and 6 days after inoculation compared to plants from cv. TMG 132. In conclusion, the results of this study demonstrated that the defence‐related enzyme activities increased upon C. cassiicola infection, regardless of the basal level of resistance of the cultivar studied. The increases in PPO activity and concentrations of TSP and LTGA derivatives, but lower LOX activity, at early stages of C. cassiicola infection were highly associated with soybean resistance to target spot.     

亚精胺诱导黄瓜幼苗对白粉病抗性的研究

以黄瓜品种‘长春密刺’幼苗为材料,研究了亚精氨(Spd)诱导黄瓜幼苗对白粉病的抗性,并测定Spd处理和白粉菌接种对黄瓜叶片4种防御酶活性及3种防卫基因表达的影响。结果显示:(1)0.2～1.0mmol.L-1 Spd对黄瓜幼苗白粉病抗性均有不同程度的诱抗效果,并以0.8mmol.L-1 Spd处理效果最明显,诱导效率可达55.3%。(2)喷施Spd或接种白粉菌均可提高黄瓜叶片过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、几丁质酶和β-1,3-葡聚糖酶的活性,且诱导并接种处理的植株叶片上述酶活性均比只诱导不接种处理的上升速度更快;同时,Spd处理和接种白粉菌可以提高植株叶片中POX、PAL、PR-1a基因的表达量。研究表明,Spd处理可以诱导防卫基因表达的增强,提高防御酶活性,显著降低病情指数,增强黄瓜幼苗对白粉病的抗性。     

Induced defence responses and protective effects on tomato against Xanthomonas vesicatoria by an aqueous extract from Solanum lycocarpum infected with Crinipellis perniciosa

This study investigated the induced defence responses and protective effects on susceptible tomato (Lycopersicon esculentum Mill.) against Xanthomonas vesicatoria (Doidge) by a heat-treated aqueous extract (VLA) from dry necrotic tissue of ‘Lobeira’ (Solanum lycocarpum St. Hil.) branches infected with the fungus Crinipellis perniciosa (Stahel) compared with acibenzolar-S-methyl (ASM), a commercial inducer of resistance. Plantlets were sprayed with VLA and ASM and challenged 4 days later with a virulent strain of X. vesicatoria, under greenhouse conditions. The disease severity, fresh weight of shoots, the activities of phenol peroxidase (POX), polyphenol oxidase (PPO), chitinase (CHI), phenylalanine ammonia-lyase (PAL), lignin deposition, and soluble phenolic contents were evaluated in the leaf tissues. Reduction of the bacterial spot severity was observed in plantlets treated with VLA which conferred 63% of the ASM protection. This protective effect and lesion reduction promoted by VLA were probably associated particularly with POX and PAL activities, lignin deposition on leaf tissues and, to a less extent, CHI activity.     

Effects of Acibenzolar-S-methyl on the specific activities of peroxidase, chitinase and phenylalanine ammonia-lyase and phenolic content of host leaves in cucumber-powdery mildew interaction

One of the properties of systemic acquired resistance in plants is its concomitance with the biochemical changes including enhancement of activities of defense-related enzymes. In this study, the effects of Acibenzolar-S-methyl (ACI) on the some of defense responses of the cucumber plants (healthy or inoculated with spore suspension of Podosphaerafusca, the causal agent of cucumber powdery mildew) were surveyed via in vivo tests. Changes of defense responses in ACI-treated cucumber plants, inoculated with pathogen or not, were studied and compared with those of non-treated control plants. Results indicated that specific activity of peroxidase increased significantly in treated plants. Increase in enzyme activity was higher in pathogen-inoculated than non-inoculated plants, thus pathogen attack stress to plant plays a role in enhancement of enzyme activity. Specific activity of phenylalanine ammonialyase showed no changes in ACI-treated non-inoculated plants, but in inoculated plants it Increased due to interaction between ACI treatment and pathogen attack stress. Specific activity of chitinase increased in both inoculated and non-inoculated ACI-treated plants at 24 hours after treatment onwards, and pathogen attack stress did not affect it. Phenolic content of ACI-treated plant tissues, despite of small fluctuations, did not show any definite pattern of changes.     

Biochemical responses associated with resistance to bacterial stalk rot caused by Dickeya zeae in maize

Bacterial stalk rot (BSR) of maize caused by Dickeya zeae is an important disease in northwest region of India. In the current study, eighty maize lines were evaluated for resistance against this disease. Of these, 20 were moderately resistant, 25 were moderately susceptible and the rest were highly susceptible to BSR. Six lines from each set were randomly selected. Activities of three antioxidant enzymes, viz. phenylalanine ammonia lyase (PAL), peroxidase (POX) and polyphenol oxidase (PPO) were analysed from these three sets of maize lines representing different levels of resistance. A trend of elevated activity of PAL, POX and PPO was observed in all the three sets of maize lines. The results showed significantly more activity of these three enzymes in moderately resistant than highly susceptible maize lines. The activity of PAL and PPO peaked after 48 hr and of POX after 72 hr of challenge inoculation by D. zeae in all the maize lines. The activity of these enzymes further correlated negatively with disease development. Our results show that PAL, POX and PPO play an important role in contributing towards resistance in maize against BSR.     

Biochemical alterations in leaves of resistant and susceptible cotton genotypes infected systemically by cotton leaf curl Burewala virus

Leaf curl disease caused by Cotton Leaf Curl Burewala virus (CLCuBuV) has been recognized as serious threat to cotton in Indian subcontinent. However, information about cotton–CLCuBuV interaction is still limited. In this study, the level of phenolic compounds, total soluble proteins, and malondialdehyde (MDA) and the activities of phenylalanine ammonia-lyase (PAL), peroxidase (POX), catalase (CAT), proteases, superoxide dismutase (SOD), and polyphenol oxidase (PPO) were studied in leaves of two susceptible (CIM-496 & NIAB-111) and two resistant (Ravi and Co Tiep Khac) cotton genotypes. Disease symptoms were mild in the resistant genotypes but were severe in highly susceptible genotypes. The results showed that phenolic compounds, proteins, PAL, POX, CAT, proteases, SOD, PPO, and MDA play an active role in disease resistance against CLCuBuV. The amount of total phenols, proteases, MDA, and PPO was significantly higher in leaves of CLCuBuV-inoculated plants of both resistant genotypes as in non-inoculated plants, and decreased in CLCuBuV-inoculated plants of both susceptible genotypes over their healthy plants. POX, protein content, SOD, and PAL activities showed lower values in resistant genotypes, while they decreased significantly in susceptible genotypes as compared to the noninoculated plants except PAL, which showed non-significant decrease. CAT was found to be increased in both susceptible and resistant genotypes with maximum percent increase in resistant genotype Ravi, as compared to non-inoculated plants. The results showed significantly higher concentrations of total phenols and higher activity of protease, MDA, SOD, and PPO in resistant genotype Ravi after infection with CLCuBuV, suggesting that there is a correlation between constitutive induced levels of these enzymes and plant resistance that could be considered as biochemical markers for studying plant-virus compatible and incompatible interactions.     

UV-B对不同发育时期离体蓝莓主要果实品质及相关酶活性的影响

该研究以幼果期、白果期、转色期的离体‘北陆’蓝莓果实为试材,设置0(CK)、5、10、15min紫外光辐照处理,24h后取样分析蓝莓果实中可溶性糖、总酚、类黄酮和花青苷含量,以及苯丙氨酸裂解酶(PAL)和查尔酮异构酶(CHI)活性的变化,探究UV-B紫外照射处理对不同发育时期蓝莓主要果实品质及相关酶活的影响。结果显示:(1)对于幼果期蓝莓,5min UV-B处理可显著增加果实内可溶性糖含量;10min UV-B处理果实PAL活性增加效果最为显著;15min UV-B处理对果实总酚和花青苷积累的促进作用最大,但显著降低了类黄酮含量和CHI活性。(2)对于白果期蓝莓,5min UV-B处理显著增加了果实类黄酮含量和CHI活性,10min处理使果实可溶性糖和总酚含量较对照分别增加25%和18%;15min处理对果实花青苷含量和PAL活性影响作用最大。(3)对于转色期蓝莓,各处理除果实可溶性糖及类黄酮含量降低外,其余物质含量均显著增加。(4)UV-B处理并未改变果实发育过程中可溶性糖、总酚、类黄酮和花青苷含量及PAL、CHI酶活性的积累规律。(5)蓝莓果内PAL活性与其可溶性糖、总酚和类黄酮的积累呈极显著正相关关系,而CHI活性仅与其可溶性糖呈极显著正相关。研究表明,UV-B辐照处理促进了幼果期和白果期可溶性糖的积累,也能促进不同发育时期蓝莓果实总酚和花青苷及白果期类黄酮的积累,对蓝莓果实主要品质能够产生积极的影响。     

杂交稻抗感白叶枯病与活性氧伤害及防御酶系统的关系

以对白叶枯病抗感不同的三个杂交稻为材料,在三叶期接种白叶枯病强毒菌株７６—２５,结果表明,杂交稻接种白叶枯病菌７２ｈ后,苯丙氨酸解氨酶（ＰＡＬ）活性和过氧化物酶（ＰＯＸ）活性升高,超氧化物歧化酶（ＳＯＤ）活性下降,膜脂过氧化产物丙二醛（ＭＤＡ）含量增加,且抗病组合的变化幅度大于感病组合,说明杂交稻抗白叶枯病与活性氧伤害及保护酶系的活性相关。     

A Vesicular Arbuscular Mycorrhizal Fungus (Glomus intraradix) Induces a Defense Response in Alfalfa Roots

Flavonoid accumulation and activities of phenylalanine ammonia-lyase (PAL), chalcone isomerase (CHI), and chitinase were followed during early colonization of alfalfa roots (Medicago sativa L. cv Gilboa) by vesicular arbuscular (VA) fungi (Glomus intraradix). Formononetin was the only flavonoid detected that showed a consistent increase in the inoculated roots. This increase depended only on the presence of the fungus in the plant rhizosphere; no colonization of the root tissue was required. CHI and chitinase activities increased in inoculated roots prior to colonization, whereas the increase in PAL activity coincided with colonization. After reaching a maximum, activities of all enzymes declined to below those of uninoculated roots. PAL inactivation was not caused by a soluble inhibitor. Our results indicate that VA fungi initiate a host defense response in alfalfa roots, which is subsequently suppressed.     

Induction of total phenolics and defence-related enzymes during β-aminobutyric acid-induced resistance in Brassica carinata against Alternaria blight

Exogenous foliar application of β-aminobutyric acid (BABA) led to a significant reduction in disease severity in Brassica carinata caused by Alternaria brassicae. To get a better insight about changes in defence-related enzymes like phenylalanine ammonia lyase (PAL) and polyphenol oxidase (PPO), isoform analysis of superoxide dismutase (SOD) and peroxidase (POX) were studied. BABA-treated plants showed a significant increase in PAL, PPO enzyme activities and total phenolic content in response to pathogen inoculation. However, isoform analysis of SOD and POX revealed no change in isoform number but a quantitative change in activity was observed in response to pathogen.     

粉红珙桐叶片呈色相关生理特性的季节变化

以四川省珙县王家镇同一生境下不同季节的粉红与绿叶珙桐叶片为试材,对其色素含量、渗透调节物质含量及相关酶活性进行测定,分析其季节性变化规律,从生理角度探寻影响珙桐叶片呈色的关键因素,以期为粉红珙桐的选育和合理栽培提供理论依据和技术参考。结果显示:(1)粉红珙桐叶片花色苷含量在春夏秋季均极显著高于绿叶珙桐,在夏季时含量最低;粉红珙桐叶片总叶绿素含量在夏季和秋季极显著低于绿叶珙桐,从春季过渡至秋季过程中逐渐上升;两者的类胡萝卜素和类黄酮含量差异不明显且在季节间较平稳。(2)粉红珙桐叶片的可溶性糖和可溶性蛋白含量略高于绿叶珙桐,夏季时达到最高;其脯氨酸含量也高于绿叶珙桐,且从春季到秋季缓慢上升。(3)粉红珙桐叶片苯丙氨酸解氨酶活性(PAL)、苯基苯乙烯酮黄烷酮异构酶(CHI)和超氧化物歧化酶活性(SOD)都显著高于绿叶珙桐,过氧化物酶(POD)活性则相反;从春季到秋季过程中,两种珙桐叶片PAL活性先减弱后增强,其POD和SOD活性则先增强后减弱,但粉红珙桐CHI活性逐渐减弱,绿叶CHI活性则先上升后下降。(4)粉红珙桐叶片花色苷含量与叶绿素含量、可溶性蛋白含量以及CHI、POD和SOD活性呈显著负相关关系。研究表明,叶片花色苷含量的增加和叶绿素含量的减少是粉红珙桐呈色的决定因素,CHI、SOD和POD活性是调节花色苷降解速率的关键酶。     

Ultrastructure of Tobacco Ringspot Virus-Induced Local Lesions in Lima Bean Leaves

Lignin is hypothesized to be a factor in the ability of cucurbits to resist fungal infection. To determine if lignin was present around sites of infection, susceptible cucumber plants and cucumber plants with systemic induced resistance were inoculated with Colletotrichum lagenarium, and the ultrastructure of plant and pathogen was examined 24 to 72 hours later. Conidia produced germ tubes with appressoria within 24 hours of inoculation on both control and induced plants. Penetration of plant epidermal cell walls occurred by 48 hours, and papillae formed at the site of penetration. Both elemental bromine and potassium permanganate were used to histochemically stain for lignin. Potassium permanganate, used together with energy dispersive X-ray microanalysis (EDS), indicated the presence of lignin in papillae and in electron-dense areas of plant cell walls directly beneath the appressoria. Using EDS, silicon was shown to be localized in the electron-dense areas of cucumber leaf cells. This is the first report of silicon in induced cucumber plants. There were no qualitative differences between the resistance reactions of induced and control plants.     

Effect of chitin hexamer and thuricin 17 on lignification-related and antioxidative enzymes in Soybean Plants

Inducers of disease resistance in crop plants have a role in sustainable agriculture. We describe a set of bacteriocins that can potentially improve plant growth by controlling specific pathogens and inducing generalized resistance. Solutions of the bacteriocin thuricin 17 and/or a chitin hexamer (a known inducer and positive control) were applied to leaves of two-week-old soybean plants, and levels of lignification-related and antioxidative enzymes were monitored. Phenyl ammonia lyase (PAL) activity in thuricin 17-treated leaves was highest at 60 h after treatment, being 61.8% greater than the control. PAL activity also was increased 18.1% at 72 h after treatment with the chitin hexamer. Tyrosine ammonia lyase (TAL) activity in leaves was 57.0% higher than the control at 48 h after treatment with thuricin 17, while such activity in chitin hexamer-treated leaves was increased by 23.8% at 72 h. At 36 h after treatment with the chitin hexamer or chitin hexamer + thuricin 17, the total concentration of phenolic compounds was 15.3 or 19.3%, respectively, greater than the control. At 72 h, total phenolic concentrations increased by 23.2 and 19%, respectively, in response to thuricin 17 and chitin hexamer + thuricin 17. POD activity in thuricin 17-treated leaves increased by 74.6 and 81.2% at 48 and 72 h, respectively, whereas SOD activity increased by 24.9 and 79.9%, respectively, in chitin hexamer- and thuricin 17-treated leaves at 48 h. A peroxidase isozyme (31 kDa isomer) was induced in thuricin 17-treated leaves at 60 h, while catalase (59 kDa isomer) was induced in chitin hexamer-treated leaves. PAGE showed that two major SOD bands (Fe-SODs) were produced by both types of treatment. Collectively, these results indicate that the bacteriocin thuricin 17 can act as an inducer of plant disease defenses (i.e., activated lignification-related enzymes, antioxidative enzymes, and related isozymes) and that this induction is similar, but not identical, to that of the chitin hexamer elicitor. Although treatment with thuricin 17 + chitin hexamer also induced those responses, it did not present a clear pattern of additivity or synergy.     

Salicylic Acid Enhances Resistance to Fusarium oxysporum f. sp. phaseoli in Common Beans (Phaseolus vulgaris L.)

Fusarium wilt caused by Fusarium oxysporum f. sp. phaseoli (Fop) is one of the most serious diseases of common bean (Phaseolus vulgaris L.) and is especially prevalent in China. In this study, we demonstrated that exogenous application of 2 mM salicylic acid (SA) by leaf spraying could induce resistance against Fop in common beans. Accumulation of free and conjugated SA in roots was detected by HPLC analysis and compared. After 168 h of daily SA treatment, the free SA level in roots was eight times higher than in control plants. However, the conjugated SA level reached a peak at 72 h of SA treatment, which was nine times higher than in control plants, and then sharply declined at 168 h. The activities of phenylalanine ammonia lyase (PAL, EC 4.3.1.5) and peroxidases (POX, EC 1.11.1.7) in roots were 9.4 and 6.3 times higher than in control plants after 168 h of SA treatment, respectively. H₂O₂ and O₂ ^? levels reached 2.6 and 13.6 times higher, respectively, than in the control plants at 168 h after SA treatment. Host reactions of SA-treated plant roots infected by Fop observed in microscopy included the deposition of electron-dense materials along the secondary walls. However, untreated inoculated plants showed marked cell wall degradation and total cytoplasm disorganization of root cells. These results indicated that SA applied to foliar tissue is capable of enhancing the systemic acquired resistance of common bean roots to infection by Fop.     

Thermotolerance and antioxidant systems in Agrostis stolonifera: involvement of salicylic acid, abscisic acid, calcium, hydrogen peroxide, and ethylene

This study investigated whether pre-treating plants with specific putative signaling components and heat acclimation would induce tolerance of a cool-season grass, creeping bentgrass (Agrostis stolonifera var. palustris), to subsequent heat stress and whether thermotolerance induction of those pretreatments was associated with the regulation of antioxidant regenerating enzymes. The treatments included foliar application of salicylic acid (SA), abscisic acid (ABA), calcium chloride (CaCl2), hydrogen peroxide (H2O2), 1-aminocyclopropane-1-carboxylic acid (ACC, a precursor of ethylene prior to the exposure of plants to heat stress (35 degrees C) in a growth chamber. Physiological measurements including turf quality, leaf photosynthetic rate, and levels of oxidative damage demonstrated that all treatments increased heat tolerance. The better heat tolerance for pre-treated plants as compared to controls was related to the protection of oxidative damage under heat stress. APX activity increased over the first 2 days and 5 days of heating for ACC and CaCl2 respectively, but for only 12 h for H2O2. SA and ABA pre-treatments had no effects on APX activity earlier, but maintained APX activity at a significantly higher level than in controls after 24 h of heating. SA and ABA pre-treatments had no effects on POX activity. ACC treatment significantly increased POX activity. Pre-treatment with CaCl2, H2O2, and HA reduced POX activity, particularly during the later phase of heating. Plants treated with SA, CaCl2, H2O2 and HA had lower CAT activity than their control plants prior to heating and within 48 h of heat stress. ABA and ACC pre-treatments maintained higher CAT activity than the controls after 48 h of heating. ACC, CaCl2, or HA pre-treatments increased SOD activity only before 5 days of heat stress. SA and ABA pre-treatments had less effect on APX activity earlier under heat stress. These results suggest that specific groups of potential signaling molecules may induce tolerance of creeping bentgrass to heat stress by reducing oxidative damage.     

Induction of Defence Responses in Roots and Mesocotyls of Sorghum Seedlings by Inoculation with Fusarium thapsinum and F. proliferatum, Wounding and Light

The defence reactions of sorghum seedlings 7 days after inoculation with Fusarium thapsinum and F. proliferatum, and interactions with wounding and exposure to light were studied to determine whether responses to these fungi differed from those to abiotic stresses. In non‐wounded plants, inoculation with both fungi increased concentrations of anthocyanins and soluble phenolics and activities of peroxidase (POX), chitinase and β‐1,3‐glucanase in the roots, and increased β‐1,3‐glucanase activity in the mesocotyls. There was no effect of inoculation on phenylalanine ammonia‐lyase (PAL) activity. Wounding by itself increased anthocyanin content of mesocotyls. Wounding also had a variety of interactions with inoculation. Exposure to light had very little effect on any defence response measured. A time course experiment showed that induction of chitinase and β‐1,3‐glucanase occurred in less than 24 h after inoculation. POX activity increased 2 days after inoculation, followed by a transient increase in PAL activity. The content of anthocyanins and soluble phenolics in roots of inoculated seedlings increased gradually compared with controls over 6 days. The responses of sorghum seedlings to inoculation with F. thapsinum and F. proliferatum were similar to those found by other workers following challenge by necrotrophic pathogens and were different from those induced by wounding and exposure to light.     

Induction of the activities of antioxidative enzymes and the levels of malondialdehyde in cucumber seedlings as a consequence of Bemisia tabaci (Hemiptera: Aleyrodidae) infestation

The activities of antioxidative enzymes and the concentration of malondialdehyde were assayed in cucumber leaves (Cucumis sativus L.) 0, 6, 12, 24, 48, 72, and 96 h after the cucumber seedlings were infested by Bemisia tabaci (Gennadius). The results indicated that the activities of antioxidative enzymes were increased after herbivore infestation, including superoxide dismutase (SOD), and peroxidase (POD), and the levels of malondialdehyde (MDA), which is a product of membrane lipid peroxidation in the leaves. The enzymes and MDA showed peaks of different activity levels at 24 and 48 h after the infestation. SOD activity reached the highest peak, 10.4% higher than control, at 24 h, POD activity reached the highest peak, 213.2% higher than control, at 6 h, catalase (CAT) activity was not statistically significant compared with the control, and MDA content reached the highest peak, 59.9% higher than control, at 48 h. The results suggested that the enhanced activities of antioxidative enzymes and MDA content may contribute to bioprotection of cucumber plants against B. tabaci infestation. Handling editor: Henryk Czosnek