Induction of defense-related proteins in tomato roots treated with Pseudomonas fluorescens Pf1 and Fusarium oxysporum f. sp. lycopersici

Pseudomonas fluorescens isolate Pf1 was found to protect tomato plants from wilt disease caused by Fusarium oxysporum f. sp. lycopersici. Induction of defense proteins and chemicals by P. fluorescens isolate Pf1 against challenge inoculation with F. oxysporum f. sp. lycopersici in tomato was studied. Phenolics were found to accumulate in bacterized tomato root tissues challenged with F. oxysporum f. sp. lycopersici at one day after pathogen challenge. The accumulation of phenolics reached maximum at the 5^th day after pathogen challenge. In pathogen-inoculated plants, the accumulation started at the 2^nd day and drastically decreased 4 days after the pathogen inoculation. Activities of phenylalanine ammonia-lyase (PAL), peroxidase (PO) and polyphenol oxidase (PPO) increased in bacterized tomato root tissues challenged with the pathogen at one day after pathogen challenge and activities of PAL and PO reached maximum at the 4^th day while activity of PPO reached maximum at the 5^th day after challenge inoculation. Isoform analysis revealed that a unique PPO1 isoform was induced and PO1 and PPO2 isoforms were expressed at higher levels in bacterized tomato root tissues challenge inoculated with the pathogen. Similarly, -1,3 glucanase, chitinase and thaumatin-like proteins (TLP) were induced to accumulate at higher levels at 3-5 days of challenge inoculation in bacterized plants. Western blot analysis showed that chitinase isoform Chi2 with a molecular weight of 46 kDa was newly induced due to P. fluorescens isolate Pf1 treatment challenged with the pathogen. TLP isoform with molecular weight of 33 kDa was induced not only in P. fluorescens isolate Pf1-treated root tissues challenged with the pathogen but also in roots treated with P. fluorescens isolate Pf1 alone and roots inoculated with the pathogen. These results suggest that induction of defense enzymes involved in phenylpropanoid pathway and accumulation of phenolics and PR-proteins might have contributed to restriction of invasion of F. oxysporum f. sp. lycopersici in tomato roots.     

Induction of early blight resistance in tomato by <Emphasis Type="Italic">Quercus infectoria</Emphasis> gall extract in association with accumulation of phenolics and defense-related enzymes

Treatment of tomato leaves with aqueous extract (0.5%) of the galls of Quercus infectoria significantly reduced infection from subsequent inoculation with Alternaria solani, the tomato early blight pathogen. When the leaves were challenge-inoculated with A. solani 3 d after application of Q. infectoria gall extract (QIGE), the percent defoliation decreased from 33.6 to 7.3. Two to three day pre-treatment with QIGE reduced the percent defoliation by 77 percent. The biochemical responses of tomato plants to QIGE were also studied. In tomato plants treated with QIGE, phenolic content increased rapidly, reached the maximum at 2 d after treatment. Phenylalanine ammonia-lyase (PAL) activity increased significantly from 1 d after treatment and the maximum enzyme activity was recorded 2 d after treatment at which period a 3-fold increase in PAL activity was observed when compared to the control. Peroxidase (PO) activity was also significantly increased 1 d after treatment and the maximum activity was reached 2 d after treatment. Peroxidase isozyme analysis indicated that PO-1 was increased dramatically in tomato leaves 1 d after treatment and maintained at the same level throughout the experimental period of 6 d. When tomato leaves were treated with QIGE, a two-fold increase in chitinase and β-1,3-glucanase activities was recorded 2 and 3 d respectively, after treatment. The enhanced activities of defense-related enzymes and elevated levels of phenolics in QIGE-treated tomato plants between 1 and 3 d after treatment suggest that these induced biochemical defenses may be involved in the suppression of early blight by QIGE.     

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.     

Changes in pathogenesis-related proteins in pepper plants with regard to biological control of phytophthora blight with <Emphasis Type="Italic">Paenibacillus illinoisensis</Emphasis>

To evaluate the biocontrol effectiveness of chitinase-producing bacterium, Paenibacillus illinoisensis strain KJA-424 against pathogenic strain of Phytophthora capsici in pepper plants, growth response and kinetics of pathogen related (PR) proteins were estimated after inoculation with P. capsici (P), and with a combination of P. capsici and strain KJA-424 cell culture (P+A). Fresh weight and chlorophyll content in shoots at P+A-treated plants significantly increased by 23.4 and 34.2%, respectively after 7days of inoculation, compared to P-treated plants. Root mortality in P+A-treated plants was significantly reduced compared to P-treated plants. Seven days after inoculation, the activities of -1,3-glucanase, cellulase and chitinase in P-treated roots had decreased by 54.8, 36.5 and 52.8%, respectively, compared to P+A-treated roots, while those in P-treated leaves increased by 22.8, 36.3 and 23.8%, respectively, compared to those in P+A-treated leaves. The activities of -1,3-glucanase, cellulase and chitinase in roots are negatively correlated with root mortality. All these results suggest that the inoculation of an antagonist, P. illinoisensis alleviates root mortality, reduction of PR proteins in roots, and activates of PR proteins in leaves infected by P. capsici.     

Time course study on accumulation of cell wall-bound phenolics and activities of defense enzymes in tomato roots in relation to <Emphasis Type="Italic">Fusarium</Emphasis> wilt

Major cell wall-bound phenolic compounds were detected and identified in roots of tomato at different stages of growth. Alkaline hydrolysis of the cell wall material of the root tissues yielded ferulic acid as the major bulk of the phenolic compounds. Other phenolic compounds identified were 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin and 4-coumaric acid. All the six phenolic acids were higher in very early stage of plant growth. Ferulic acid, 4-hydroxybenzoic acid and 4-coumaric acid exhibited a decreasing trend up to 60 days and then the content of these phenolic acids increased somewhat steadily towards the later stage of growth. Total phenolics, phenylalanine ammonia-lyase (PAL) activity and peroxidase (POD) activity were in tandem match with the occurrence pattern of the phenolic acids. Ferulic acid showed highest antifungal activity against tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici. The results of this study may be interpreted to seek an explanation for high susceptibility of tomato plants at flowering stage to Fusarium wilt. It may also be concluded that greater amounts of ferulic acid in combination with other phenolics and higher level of PAL and POD activities after 60 days of growth may have a role in imparting resistance against Fusarium wilt at a late stage of plant growth.     

Biological control ofFusarium oxysporum f.sp.cubense in banana by inoculation withPseudomonas fluorescens

Native strains ofPseudomonas fluorescens exhibitedin vitro antibiosis towards isolates of races 1 and 4 ofFusarium oxysporum f.sp.cubense, the Panama wilt pathogen of banana. The seedlings ofMusa balbisiana seedlings treated withP. fluorescens showed less severe wilting and internal discolouration due toF. oxysporum f.sp.cubense infection in greenhouse experiments. In addition to suppressing Panama wilt, bacterized seedlings ofM. balbisiana also showed better root growth and enhanced plant height.     

Biological control of root-knot nematode (Meloidogyne javanica) disease by Pseudomonas fluorescens (Chao)

Plant growth-promoting Rhizobacteria is currently developed as an biocontrol agent against many plant pathogens. In this research, biological control of root-knot nematode (Meloidogyne javanica) by Pseudomonas fluorescens was investigated in greenhouse and laboratory experiments. Results showed that 10⁹?(CFU/ml) of P. fluorescens decreased nematode infection and other parameters significantly, compared to the control. P. fluorescens was able to cause destruction of nematode egg mass matrix and significantly decreased nematode egg hatching level. Specific activities of resistance-related enzymes, namely peroxidase (POX) and phenylalanine ammonia lyase (PAL), increased significantly in P. fluorescens-inoculated plants. Maximum activities of POX and PAL were observed at the 5?days after inoculation, respectively. Results suggested that the destruction of eggs and plant defence mechanisms leading to systemic resistance are two main suppression mechanisms used by P. fluorescens against nematode.     

Purification,characterization and differential hormonal regulation of a β-1,3-glucanase and two chitinases from chickpea (Cicer arietinum L.)

Chickpea (Cicer arietinum L.) cell-suspension cultures were used to isolate one -1,3-glucanase (EC 3.2.1.29) and two chitinases (EC 3.2.1.14). The -1,3-glucanase (M_r = 36 kDa) and one of the chitinases (M_r = 32 kDa) belong to class I hydrolases with basic isoelectric points (10.5 and 8.5, respectively) and were located intracellularly. The basic chitinase (BC) was also found in the culture medium. The second chitinase (M_r = 28 kDa), with an acidic isoelectric point of 5.7, showed homology to N-terminal sequences of class III chitinases and represented the main protein accumulating in the culture medium. Polyclonal antibodies raised against the basic -1,3-glucanase (BG) and the acidic chitinase (AC) were shown to be monospecific. The anti-AC antiserum failed to recognize the BC on immune blots, confirming the structural diversity between class I and class III chitinases. Neither chitinase exhibitied lysozyme activity. All hydrolases were endo in action on appropriate substrates. The BC inhibited the hyphal growth of several test fungi, whereas the AC failed to show any inhibitory activity. Expression of BG activity appeared to be regulated by auxin in the cell culture and in the intact plant. In contrast, the expression of neither chitinase was apparently influenced by auxin, indicating a differential hormonal regulation of -1,3-glucanase and chitinase activities in chickpea. After elicitation of cell cultures or infection of chickpea plants with Ascochyta rabiei, both system were found to have hydrolase patterns which were qualitatively and quantitatively comparable. Finally, resitant (ILC 3279) and susceptible (ILC 1929) cultivars of chickpea showed no appreciable differences with regard to the time and amount of hydrolase accumulation after inoculation with spores of A. rabiei.Abbreviations AC acidic chitinase - BC basic chitinase - BG = basic -1,3-glucanase - CM-Chitin-RBV carboxymethylated-chitin-remazol brilliant violet - 2,4-D 2,4-dichlorophenoxyacetic acid - ILC international legume chickpea - M_r relative molecular mass - pI isoelectric point - SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis We thank the Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie for financial support and ICARDA, Aleppo, Syria, for the provision of seed material. We also thank Dr. B. Fritig (Institut de Biologie Moléculaire des Plantes, CNRS, Straßbourg, France) and Dr. F. Meins, Jr. (Friedrich-Miescher-Institut, Basel, Switzerland) for their kind gifts of antibodies.     

Interaction of Fusarium Oxysporum f.sp. Cubense with Pseudomonas Fluorescens Precolonized to Banana Roots

Effect of precolonization of banana cv Neeypovan roots with Pseudomonas fluorescens on infection with Fusarium oxysporum f.sp. cubense was studied. Under in vitro conditions Pseudomonas fluorescens clearly inhibited Fusarium oxysporum f.sp. cubense. Fluorescein isothiocyanate-tagged antibodies raised in a rabbit system for Pseudomonas fluorescens and Fusarium oxysporum f.sp. cubense separately were used to study the spread of both organisms in banana root. It was observed that precolonization with Pseudomonas fluorescens could reduce Fusarium oxysporum f.sp. cubense colonization by 72%, and also correlated with a number of structural changes in the cortical cells, mainly with densely stained amorphous material and polymorphic wall thickenings as revealed by light and electron microscopic studies. Massive depositions of unusual structures at sites of fungal entry was also noticed, which clearly indicated that bacterized root cells were signalled to mobilize a number of defence structures for preventing the spread of pathogen in the tissue. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Primary structure and expression of mRNAs encoding basic chitinase and 1,3-β-glucanase in potato

Infection of potato leaves (Solanum tuberosum L. cv. Datura) by the late blight fungus Phytophthora infestans, or treatment with fungal elicitor leads to a strong increase in chitinase and 1,3--glucanase activities. Both enzymes have been implicated in the plant's defence against potential pathogens. In an effort to characterize the corresponding genes, we isolated complementary DNAs encoding the basic forms (class I) of both chitinase and 1,3--glucanase, which are the most abundant isoforms in infected leaves. Sequence analysis revealed that at least four genes each are expressed in elicitor-treated leaves. The structural features of the potato chitinases include a hydrophobic signal peptide at the N-terminus, a hevein domain which is characteristic of class I chitinases, a proline- and glycine-rich linker region which varies among all potato chitinases, a catalytic domain, and a C-terminal extension. The potato 1,3--glucanases also contain a N-terminal hydrophobic signal peptide and a C-terminal extension, the latter comprising a potential glycosylation site. RNA blot hybridization experiments showed that basic chitinase and 1,3--glucanase are strongly and coordinately induced in leaves in response to infection, elicitor treatment, ethylene treatment, or wounding. In addition to their activation by stress, both types of genes are regulated by endogenous factors in a developmental and organ-specific manner. Appreciable amounts of chitinase and 1,3--glucanase mRNAs were found in old leaves, stems, and roots, as well as in sepals of healthy, untreated plants, whereas tubers, root tips, and all other flower organs (petals, stamen, carpels) contained very low levels of both mRNAs. In young leaves and stems, chitinase and 1,3--glucanase were differentially expressed. While chitinase mRNA was abundant in these parts of the plant, 1,3--glucanase mRNA was absent. DNA blot analysis indicated that in potato, chitinase and 1,3--glucanase are encoded by gene families of considerable complexity.     

Biological control of Radopholus similis in banana by combined application of the mutualistic endophyte Fusarium oxysporum strain 162, the egg pathogen Paecilomyces lilacinus strain 251 and the antagonistic bacteria Bacillus firmus

The biological control efficacy of single or multiple applications of the mutualistic endophyte Fusarium oxysporum strain 162, the egg pathogen Paecilomyces lilacinus strain 251 and the antagonistic bacteria Bacillus firmus toward Radopholus similis was investigated in pot trials with banana under glasshouse conditions. R. similis was controlled substantially in single and combined applications of F. oxysporum with P. lilacinus or B. firmus. The combination of F. oxysporum and P. lilacinus caused a 68.5% reduction in nematode density whereas the individual applications reduced the density by 27.8% and 54.8% over the controls, respectively. Combined application of F. oxysporum and B. firmus was the most effective treatment in controlling R. similis on banana (86.2%), followed by B. firmus alone (63.7%). The compatibility of the biocontrol agents, as well the capacity of F. oxysporum to colonize banana roots in the absence or presence of P. lilacinus was also investigated. P. lilacinus did not adversely affect endophytic colonization by F. oxysporum. Biological control of R. similis in banana can therefore be enhanced via combined applications of antagonists with different modes of action that target different stages in the infection process. Handling editor: Ralf-Udo Ehlers     

18份广东香蕉种质对枯萎病的抗性评价

【背景】香蕉枯萎病是世界性的香蕉毁灭性病害,尚无有效药剂防控,筛选抗病品种是目前理想的防治方法。【方法】采用组培苗伤根接种法,研究了18份香蕉种质对香蕉枯萎病菌4号生理小种的抗性水平,并根据病情指数进行抗性分级。【结果】在供试的18份香蕉种质中,2份(东莞大蕉、抗枯5号)高抗,2份(碧盛、大丰)抗病,3份(抗枯1号、粉杂、农科1号)中抗,7份(粤优抗1号、广东-741、泰国B9、大蕉、台湾8号、海贡蕉、威廉斯8818)感病,4份(巴西、广东2号、广粉1号、粉蕉)高感。【结论与意义】不同香蕉种质对香蕉枯萎病菌4号生理小种的抗病性存在较大差异,本研究初步筛选出7份抗枯萎病的香蕉种质,为香蕉枯萎病抗病育种提供了依据,为病区种植香蕉品种提供了有效参考。     

Induction of defence-related enzymes in susceptible variety of banana: role of Fusarium-derived elicitors

Elicitor prepared from the Fusarium oxysporum f. sp cubense (Foc) isolated from infected banana rhizosphere induced the accumulation of resistance-associated enzymes in leaves of susceptible and resistant variety of banana. Roots of Grand Naine (susceptible) and robusta (resistant) variety were inoculated with 1 g/l Foc elicitors. Distinct difference in peroxidase, polyphenol oxidase, β-1,3-glucanase, chitinase and phenolics was observed in control plants of resistant and susceptible varieties. Induced defence-related enzymes in susceptible variety were increased tothe level of untreated resistant variety. This depicted that Fusarium-derived elicitor effectively induced defence in susceptible variety to the apparent level of untreated resistant variety.     

Endophytic Pseudomonas fluorescens Endo2 and Endo35 induce resistance in black gram (Vigna mungo L. Hepper) to the pathogen Macrophomina phaseolina

Abstract

The effect of endophytic Pseudomonas fluorescens isolates Endo2 and Endo35 on induced systemic disease protection against dry root rot of black gram (Vigna mungo L. Hepper) caused by Macrophomina phaseolina was investigated under glasshouse conditions. When the bacterized black gram plants were inoculated with dry root rot pathogen, the activities of peroxidase (PO), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL) were stimulated in addition to accumulation of phenolics and lignin. Activity of phenylalanine ammonia-lyase (PAL) reached the maximum 24 h after pathogen challenge inoculation, whereas the activities of PO and PPO reached the maximum at 72 h and 48 h, respectively. Isoform analysis revealed that a unique PPO3 isozyme was induced in bacterized black gram tissues inoculated with the pathogen. Phenolics were found to accumulate in bacterized black gram tissues challenged with M. phaseolina one day after pathogen challenge. The accumulation of phenolics reached maximum at the third day after pathogen inoculation. Similar observation was found in the lignin content of black gram plants. In untreated control plants, the accumulation of defence enzymes and chemicals started at the first day and drastically decreased 3 days after pathogen inoculation. These results suggest that induction of defense enzymes involved in phenylpropanoid pathway and accumulation of phenolics and PR-proteins might have contributed to restricting invasion of Macrophomina phaseolina in black gram roots.     

Correlations between Accumulation of Dianthramides, Dianthalexin and Unknown Compounds, and Partial Resistance to Fusarium oxysporum f. sp. dianthi in Eleven Carnation Cultivars

The accumulation of dianthramides, dianthalexin and other phenolic compounds in carnation cells at different times before and after inoculation of plants with Fusarium oxysporum f. sp. dianthi, and the development of wilt symptoms were compared for eleven carnation cultivars. Untreated and water-treated controls contained only a little acetone-extractable phenolics. Upon inoculation with F. oxysporum f. sp dianthi, all cultivars accumulated the same range of phenolic compounds, including several dianthramides and dianthalexin, but in different proportions. The total amount of accumulated phenolic compounds per fresh material weight of the extracted stem segments could not be related to the disease resistance level of the different cultivars. However, the accumulation of dianthramides and dianthalexin in the cultivars was positively correlated to resistance, while the accumulation of two other, as yet unidentified, compounds was inversely related to resistance.     

Acibenzolar-S-methyl induced resistance to <Emphasis Type="Italic">Phytophthora capsici</Emphasis> in pepper leaves

The leaves of pepper (Capsicum anuum L.) were inoculated with Phytophthora capsici Leonian 3 d after treatment with acibenzolar-S-methylbenzo [1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester (ASM) and resistance to Phytophthora blight disease was investigated. Results showed that P. capsici was significantly inhibited by ASM treatment by up to 45 % in planta. The pepper plants responded to ASM treatments by rapid and transient induction of L-phenylalanine ammonia-lyase (PAL), increase in total phenol content and activities of chitinase and β-1,3-glucanase. No significant increases in enzyme activities were observed in water-treated control plants compared with the ASM-treated plants. Therefore it may be suggested that ASM induces defense-related enzymes, PAL activity, PR proteins and phenol accumulation in ASM-treated plants and contribute to enhance resistance against P. capsici.