Effects of Pseudomonas aureofaciens 63-28 on defense responses in soybean plants infected by Rhizoctonia solani

The objective of this work was to investigate the ability of the plant growth-promoting rhizobacterium Pseudomonas aureofaciens 63-28 to induce plant defense systems, including defense-related enzyme levels and expression of defense-related isoenzymes, and isoflavone production, leading to improved resistance to the phytopathogen Rhizoctonia solani AG-4 in soybean seedlings. Seven-dayold soybean seedlings were inoculated with P. aureofaciens 63-28, R. solani AG-4, or P. aureofaciens 63-28 plus R. solani AG-4 (P+R), or not inoculated (control). After 7 days of incubation, roots treated with R. solani AG-4 had obvious damping-off symptoms, but P+R-treated soybean plants had less disease development, indicating suppression of R. solani AG-4 in soybean seedlings. Superoxide dismutase (SOD) and catalase (CAT) activities of R. solani AG-4-treated roots increased by 24.6% and 54.0%, respectively, compared with control roots. Ascorbate peroxidase (APX) and phenylalanine ammonia lyase (PAL) activities of R. solani AG-4-treated roots were increased by 75.1% and 23.6%, respectively. Polyphenol oxidase (PPO) activity in soybean roots challenged with P. aureofaciens 63-28 and P+R increased by 25.0% and 11.6%, respectively. Mn-SOD (S1 band on gel) and Fe-SOD (S2) were strongly induced in P+R-treated roots, whereas one CAT (C1) and one APX (A3) were strongly induced in R. solani AG-4- treated roots. The total isoflavone concentration in P+Rtreated shoots was 27.2% greater than the control treatment. The isoflavone yield of R. solani AG-4-treated shoots was 60.9% less than the control.     

施硅增强水稻对纹枯病的抗性

采用水培的方法,从细胞学和生理生化方面研究了硅增强水稻对纹枯病的抗性作用。结果表明：加硅处理的水稻叶片硅化细胞和叶片表面的硅元素含量均显著高于缺硅处理（对照）：接种纹枯病菌后,加硅处理的MDA含量总体上低于缺硅处理,峰值尤为显著;加硅处理的SOD活性始终高于缺硅处理,接种后第4天加硅处理SOD活性较低时,其POD活性较高,而缺硅处理的POD活性较低,表明硅增强了SOD和POD之间的协调性;接种后硅对CAT和PAL活性没有产生明显影响,但降低了PP0活性;加硅能显著降低水稻植株的纹枯病病情指数。     

Expression patterns of chitinase produced from Paenibacillus chitinolyticus with different two culture media

To investigate the expression patterns of chitinase isozymes on native-PAGE and SDS-PAGE gels Paenibacillus chitinolyticus MP-306 was cultured on culture media with and without chitin substrate. P. chitinolyticus MP-306 had a strong chitinolytic activity on colloidal chitin medium. Chitinase isozymes of MP-306 were expressed as six bands (CN1-CN6) on native-PAGE gels and thirteen bands (CS1-CS13) on SDS-PAGE gels after incubation in chitin medium. Three bands (CN1, CN2, and CN3) of chitinase isozymes of MP-306 on native-PAGE gels were expressed as nine bands (CS1, CS2, CS3, CS4, CS5, CS6, CS8, CS10, and CS13) of chitinase isozymes on SDS-PAGE gels. Three bands (CN4, CN5, and CN6) of chitinase isozymes of MP-306 were strongly inhibited by metal ions on native-PAGE and SDS-PAGE gels.     

Impact of atmospheric CO2 on growth, photosynthesis and nitrogen metabolism in cucumber (Cucumis sativus L.) plants

Expression and activity of nitrate reductase (NR; EC 1.6.6.1) and glutamine synthetase (GS; EC 6.3.1.2) were analysed in relation to the rate of CO(2) assimilation in cucumber (Cucumis sativus L.) leaves. Intact plants were exposed to different atmospheric CO(2) concentrations (100, 400 and 1200microLL(-1)) for 14 days. A correlation between the in vivo rates of net CO(2) assimilation and the atmospheric CO(2) concentrations was observed. Transpiration rate and stomatal conductance remained unaffected by CO(2) levels. The exposure of the cucumber plants to rising CO(2) concentrations led to a concomitant increase in the contents of starch and soluble sugars, and a decrease in the nitrate content in leaves. At very low CO(2), NR and GS expression decreased, in spite of high nitrate contents, whereas at normal and elevated CO(2) expression and activity were high although the nitrate content was very low. Thus, in cucumber, NR and GS expression appear to be dominated by sugar levels, rather than by nitrate contents.     

The role of raffinose in the cold acclimation response of Arabidopsis thaliana

In many plants raffinose family oligosaccharides are accumulated during cold acclimation. The contribution of raffinose accumulation to freezing tolerance is not clear. Here, we investigated whether synthesis of raffinose is an essential component for acquiring frost tolerance. We created transgenic lines of Arabidopsis thaliana accessions Columbia-0 and Cape Verde Islands constitutively overexpressing a galactinol synthase (GS) gene from cucumber. GS overexpressing lines contained up to 20 times as much raffinose as the respective wild-type under non-acclimated conditions and up to 2.3 times more after 14 days of cold acclimation at 4 degrees C. Furthermore, we used a mutant carrying a knockout of the endogenous raffinose synthase (RS) gene. Raffinose was completely absent in this mutant. However, neither the freezing tolerance of non-acclimated leaves, nor their ability to cold acclimate were influenced in the RS mutant or in the GS overexpressing lines. We conclude that raffinose is not essential for basic freezing tolerance or for cold acclimation of A. thaliana.     

Toxicity of chitinase-producing Bacillus thuringiensis ssp. kurstaki HD-1 (G) toward Plutella xylostella

One-hundred fifty isolates of Bacillus thuringiensis were tested for their ability to produce chitinase using colloidal chitin agar as the primary plating medium. Of 14 strains that produced chitinase, B. thuringiensis ssp. kurstaki HD-1(G) was identified as the highest chitinase producer and selected for further study. This bacterium produced the highest amount of chitinase (19.3 mU/ml) when it was cultivated in nutrient broth supplemented with 0.3% colloidal chitin on a rotary shaker (200 rpm) at 30 degrees C for 2 days. The toxicities of B. thuringiensis ssp. kurstaki HD-1(G) and B. thuringiensis ssp. kurstaki wa-p-2, a chitinase nonproducer, were assayed toward Plutella xylostella (diamondback moth) larvae, resulting in LC(50)'s of 4.93 x 10(4) and 1.32 x 10(5) spores/ml, respectively. If the culture broth from B. thuringiensis ssp. kurstaki HD-1(G) was used as the suspending liquid instead of phosphate buffer, their LC(50)'s were reduced to 6.23 x 10(3) and 7.60 x 10(4) spores/ml, respectively. The histopathological changes of the midgut epithelial cells of diamondback moth larvae were compared after feeding on B. thuringiensis ssp. kurstaki HD-1(G) with and without the presence of supernatant containing chitinase under light microscopy and transmission electron microscopy. The midgut epithelial cells of larvae fed for 30 min in the presence of chitinase, with or without spores and endotoxin crystals, appeared more elongated and swollen than those of the control larvae. A number of different cellular changes such as extensive cellular disintegration and appearance of numerous vacuoles were observed from the larvae fed on B. thuringiensis ssp. kurstaki HD-1(G) supplemented with supernatant containing chitinase. Thus increased toxicity and changes in epithelial cells were correlated with the presence of chitinase but this was not distinguished from the possible presence of vegetative-stage insecticidal proteins.     

Expression Patterns of PR proteins with Different Extract Methods during Germination of Rape Seed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

This study was conducted to investigate the expression patterns of pathogenesis-related proteins (chitinase, β-1,3-glucanase and peroxidase) using activity staining of native-polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE during germination of rape seed (Brassica napus L. cv. Saturnin). The crude enzymes were extracted by distilled water (DW, pH 6.0) and 100 mM K-PO₄ buffer (pH 7.0). The expression patterns of chitinase isozymes changed clearly on 10% native-PAGE gel with DW and K-PO₄ buffer extract and on 12% SDS-PAGE gel with K-PO₄ buffer extract, except for 12% SDS-PAGE conducted using DW during germination. The active bands of the chitinase isozymes were observed as four major bands (ch1, ch2, 86, and 78 kDa) and three minor bands (71, 60, and 54 kDa) on 10% native-PAGE gel conducted using DW and K-PO₄ buffer extract. The two active bands on the 12% (w/v) SDS-PAGE gel presented as 34 and 29 kDa with DW extract, whereas one active band of 34 kDa was observed when the K-PO₄ buffer extract was used. Active bands of β-1,3-glucanase isozymes changed slightly on 10% native-PAGE gel with DW and K-PO₄ buffer extract during germination. The active band of β-1,3-glucanase isozymes were shown to have a high molecular weight (G1 and G2) on native-PAGE gel with DW extract at 0, 1, 2, and 3 days after germination, but not at 4 and 5 days. One active band of β-1,3-glucanase presented as G1 in the K-PO₄ buffer extract. Active staining of peroxidase was stronger earlier in the DW extract than K-PO₄ buffer extract at 2 days. The active bands showed as P1 and P2 in both DW and K-PO₄ buffer extract at 5 days after germination.     

Chitinolytic activities in Bacillus thuringiensis and their synergistic effects on larvicidal activity

AIMS: To investigate the distribution of chitinase in Bacillus thuringiensis strains, and the enhancing effects of the chitinase-producing B. thuringiensis strains on insecticidal toxicity of active B. thuringiensis strain against Spodoptera exigua larvae. METHODS AND RESULTS: The chitinolytic activities of B.thuringiensis strains representing the 70 serotypes were investigated by the whitish opaque halo and the colorimetric method. Thirty-eight strains produced different levels of chitinase at pH 7.0, and so did 17 strains at pH 10.0. The strain T04A001 exhibited the highest production, reaching a specific activity of 355 U ml(-1) in liquid medium. SDS-PAGE and Western blotting showed that the chitinase produced by some B. thuringiensis strains had a molecular weight of about 61 kDa. The bioassay results indicated that the chitinase-producing B. thuringiensis strains could enhance the insecticidal activity of B. thuringiensis strain DL5789 against S. exigua larvae, with an enhancing ratio of 2.35-fold. CONCLUSION: This study demonstrated that chitinase was widely produced in B. thuringiensis strains and some of the strains could enhance the toxicity of active B. thuringiensis strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first investigation devoted exclusively to analyse the distribution of chitinase in B. thuringiensis. It infers that the chitinase produced by B. thuringiensis might play a role in the activity of the biopesticide.     

The role of chitosan in protection of soybean from sudden death syndrome caused by Fusarium solani f. sp. glycines

The in vitro antifungal properties of chitosan and its role in protection of soybean from a sudden death syndrome (SDS) were evaluated. Chitosan inhibited the radial and submerged growth of F. solani f. sp. glycines with a marked effect at concentrations up to 1mg/ml indicating antifungal property and at 3mg/ml was able to delay SDS symptoms expression on soybean leaves for over three days after fungal inoculation when applied preventively. Chitosan was able to induce the level of chitinase activity in soybean resulting in the retardation of SDS development in soybean leaves. However, the SDS symptoms gradually appeared and were associated with the reduction of chitinase activity level after five days of infection period. These results suggested the role of chitosan in partially protecting soybeans from F. solani f. sp. glycines infection.     

Induction of chitinase activity by exogenous cytokinins in excised dark-grown cucumber cotyledons: involvement of Ca and staurosporine-sensitive protein kinase(s) in cytokinin signaling

Cotyledons were excised from 7-day-old dark-grown cucumber seedlings and treated with water, benzyladenine (BA), kinetin (K), zeatin (Z), or zeatin riboside (ZR) in dark after endogenous cytokinin depletion. We have compared changes in chitinase (EC. 3.2.1.14) activity induced by these cytokinins. We find that the activities of chitinase and its isoforms increase by approximately 3- to 6-fold following BA, Z, and ZR treatments. Among these treatments, Z was more effective. K was totally ineffective in inducing chitinase activity. Immunoblot analysis suggests that the cytokinin Z-induction of enzyme activity is due to the induction of higher chitinase protein levels and not the activation of existing enzyme. Furthermore, the Z-induced chitinase activity and its protein accumulation were completely inhibited by the protein kinase inhibitor staurosporine, whereas the protein phosphatase inhibitor sodium fluoride was not effective in such inhibitions. Treatment of cotyledons with extemal CaCl₂ and calcium ionophore increased the basal chitinase activity by 6- and 5-fold, respectively. Moreover, the effects of staurosporine, sodium fluoride, and Ca²⁺ on Z-induced chitinase activity correlate with their effects on chitinase protein levels. Taken together, our data suggests Ca²⁺ and staurosporine-sensitive protein kinase(s) as components of the cytokinin transduction machinery involving induction of chitinase in cucumber.     

Effect of abiotic resistance inducers, γ-amino-n-butyric acid (GABA), ascorbic acid and chitosan on certain enzyme activities of eggplant inoculated with root-knot nematode,Meloidogyne incognita

Treatments with γ-amino-n-butyric acid (GABA), ascorbic acid (vitamin C) and chitosan by foliar spray or root dipping technique to eggplant growing under greenhouse conditions before and after inoculation of Meloidogyne incognita showed a generalised increase in the activity of the enzymes, peroxidase (POX), polyphenol oxidase (PPO) and chitinase as compared with the infected non treated control. The maximum increase in POX activity occurred after 10?days of nematode inoculation. The relative PPO activity with chitosan at 2500?ppm, GABA at 5000?ppm and ascorbic acid at 10?ppm using root dipping was found to be 375, 338 and 175% of control, respectively. As for PPO oxidase, the maximum activity was observed after five?days of nematode inoculation by using ascorbic acid at 10?ppm followed by GABA at 5000?ppm and chitosan at 2500?ppm by root dipping (800, 767 and 600% of control), respectively, while the highest chitinase enzyme activity (281% of control) was observed using chitosan at 2500?ppm after 10?days of inoculation.     

硝态氮对黄瓜子叶谷氨酰胺合成酶和谷氨酸脱氢酶活性的影响

在硝态氮存在或缺乏的条件下,测定了黄瓜(Cucumis sativus L．)种子萌发和子叶发育过程中子叶可溶性蛋白质含量以及谷氨酰胺合成酶(GS)和谷氨酸脱氢酶(NAD(H)-GDH)活性的变化。在子叶发育初期,无论外源氮存在与否,每对子叶可溶性蛋白质含量和GS、NADH—GDH、NAD^ -GDH活性随发育上升。在外源氮存在下,第4d后,可溶性蛋白质含量虽有所下降,但基本保持恒定;第6d后,GS和NADH—GDH活性逐渐降低,NAD^ -GDH却相反增高。但在无外源氮条件下,于第4d后,可溶性蛋白质水平以及GS、NADH—GDH和NAD^ -GDH活性都逐渐降低。在子叶发育的整个过程中,外源氮对GS和NAD^ -GDH活性有促进作用,尤其是在子叶发育的后期对NAD^ -GDH活性的促进更为明显。     

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.     

Only Specific Tobacco (Nicotiana tabacum) Chitinases and [beta]-1,3-Glucanases Exhibit Antifungal Activity

Different isoforms of chitinases and [beta]-1,3-glucanases of tobacco (Nicotiana tabacum cv Samsun NN) were tested for their antifungal activities. The class I, vacuolar chitinase and [beta]-1,3-glucanase isoforms were the most active against Fusarium solani germlings, resulting in lysis of the hyphal tips and in growth inhibition. In additon, we observed that the class I chitinase and [beta]-1,3-glucanase acted synergistically. The class II isoforms of the two hydrolases exhibited no antifungal activity. However, the class II chitinases showed limited growth inhibitory activity in combination with higher amounts of class I [beta]-1,3-glucanase. The class II [beta]-1,3-glucanases showed no inhibitory activity in any combination. In transgenic tobacco plants producing modified forms of either a class I chitinase or a class I [beta]-1,3-glucanase, or both, these proteins were targeted extracellularly. Both modified proteins lack their C-terminal propeptide, which functions as a vacuolar targeting signal. Extracellular targeting had no effect on the specific activities of the chitinase and [beta]-1,3-glucanase enzymes. Furthermore, the extracellular washing fluid (EF) from leaves of transgenic plants expressing either of the secreted class I enzymes exhibited antifungal activity on F. solani germlings in vitro comparable to that of the purified vacuolar class I proteins. Mixing EF fractions from these plants revealed synergism in inhibitory activity against F. solani; the mixed fractions exhibited inhibitory activity similar to that of EF from plants expressing both secreted enzymes.     

黄瓜叶片胞间隙几丁质酶与抗霜霉菌侵染关系的研究

采用高抗霜霉病的‘津春4号’和易感霜霉病的‘长春密刺’黄瓜的温室穴盘幼苗为材料,研究了接种霜霉菌对黄瓜叶片胞间隙几丁质酶累积变化及幼苗生长的影响.结果显示:(1)与易感品种相比,抗病品种叶片胞间隙液蛋白浓度升高快、含量高;同时几丁质酶活性上升的速度快、幅度大,于接种后第2天达到峰值,且高活性维持至第7天;(2) SDS-PAGE电泳分析发现,抗病品种有一种27 kD的酸性几丁质酶在接种后第2天迅速积累,并在接种后第4、7天呈现出较高的表达量;Western blotting的结果也证实了上述胞间隙几丁质酶积累的变化.(3)与未接种对照相比,接种处理后第4、7天,易感黄瓜幼苗鲜重、干物质积累量均出现显著降低,但在接种后第7天抗病品种的上述生长指标要显著高于易感病品种.研究表明,接种霜霉菌后,抗病黄瓜叶片胞间隙几丁质酶迅速累积且活性快速升高,以减轻霜霉病对幼苗生长的侵害,这可能是黄瓜抗霜霉菌侵染的一种防御机制.     

The effects of <Emphasis Type="Italic">β</Emphasis>-amino-butyric acid on resistance of cucumber against root-knot nematode, <Emphasis Type="Italic">Meloidogyne javanica</Emphasis>

In this study, the effects of β-amino-butyric acid (BABA) on root-knot nematode(Meloidogyne javanica) infection of cucumber and accumulation of total phenolic compounds, hydrogen peroxide and activity of some enzymes related to plant defense mechanisms, i.e., guaiacol peroxidase (GPOX), polyphenol oxidase (PPO), catalase (CAT) in cucumber roots infected with nematode were investigated. Results of this study show that treating the cucumber seedlings with the above elicitor significantly reduces the nematode infection level (the nematode galls, number of egg masses per plant and number of eggs per individual egg mass) compared to control. Additionally, treatment of cucumber roots by BABA and BABA + nematode, significantly increased peroxidase, polyphenol oxidase and catalase activities in root tissues, 1 day after nematode inoculation in comparison to nematode inoculated plants as control and sterile water-treated plants. Enzyme activities reached to a maximum level at 4, 4 and 3 days after nematode inoculation, respectively. Additionally, the amount of H₂O₂, a product of oxidative stress, was significantly increased in the BABA and BABA + nematode treatments in comparison to control. Such increases have occurred in two phases and maximum levels of it were observed at 5 days after inoculation. Inoculation of cucumber plants by BABA also significantly increased accumulation of total phenol in comparison to control and maximum level of it was observed at 7 days after nematode inoculation. The results suggest that the inhibitory effect of BABA on the root-knot nematode (M. javanica) may be related to its ability to enhance defense responses in the cucumber roots.     

Activation of a Bean Chitinase Promoter in Transgenic Tobacco Plants by Phytopathogenic Fungi

The temporal and spatial expression of a bean chitinase promoter has been investigated in response to fungal attack. Analysis of transgenic tobacco plants containing a chimeric gene composed of a 1.7-kilobase fragment carrying the chitinase 5B gene promoter fused to the coding region of the gus A gene indicated that the chitinase promoter is activated during attack by the fungal pathogens Botrytis cinerea, Rhizoctonia solani, and Sclerotium rolfsii. Although induction of [beta]-glucuronidase activity was observed in tissues that had not been exposed to these phytopathogens, the greatest induction occurred in and around the site of fungal infection. The increase in [beta]-glucuronidase activity closely paralleled the increase in endogenous tobacco chitinase activity produced in response to fungal infection. Thus, the chitinase 5B-gus A fusion gene may be used to analyze the cellular and molecular details of the activation of the host defense system during pathogen attack.     

Possible correlation between increased vigour and chitinase activity expression in tobacco

Nicotiana tabacum (tobacco) plants with several—fold increasedchitinase enzyme activity levels were obtained through transformationwith a Zea mays (maize) chitinase cDNA clone, Ch2, under thecontrol of the CaMV 35S promoter. There was a linear correlation(r=0.74, P = 0.009) between the endochitinase activity and theweight of homozygous seedlings grown in the greenhouse in someexperiments. Progeny seedlings of self-pollinated regenerants(heterozygous for Ch2) grown for 18 d in Petri plates couldbe separated into vigorous and non-vigorous categories basedon size, which were predicted to be 2n or 1,n and null, respectively,for Ch2. Testing showed that the visually estimated vigour ofthese progeny co-segregating for Ch2 and npfll generally correlatedwith resistance to kanamycin and chitinase activity. There wasalso a correlation between growth and chitinase activity insuspension cultures derived from transgenic and wild-type whenmeasured after 20 d. Twelve-day-old wild-type and transformedseedlings grown in soil under culture room conditions in closedplastic containers were visibly taller and had higher seedlingweights than their counterparts grown under mist in the greenhouse.Likewise, the chitinase activity in the cell wall-bound proteinsof the faster growing seedlings was more than double that ofthe greenhouse-grown seedlings. While these studies do not always demonstrate the correlationbetween vigour and chitinase activity, overall, they do supportthe hypothesis that chitinases have a role in plant growth. Key words: Chitinase, maize endochitinase, growth, tobacco