Influence of bacteria isolated from rice plants and rhizospheres on antibiotic production by the antagonistic bacterium <Emphasis Type="Italic">Serratia marcescens</Emphasis> strain B2

Serratia marcescens strain B2 is an antagonistic bacterium that produces the red-pigmented antibiotic prodigiosin and suppresses rice sheath blight caused by Rhizoctonia solani AG-1 IA. Rice sheath blight disease was suppressed when plants were inoculated with this bacterium an hour before pathogen inoculation but not when plants were treated 4 weeks before pathogen inoculation. In both cases the bacteria were detected in the rice rhizosphere 4 weeks after inoculation. Bacteria isolated from the rice plant and rhizosphere inhibited biosynthesis of prodigiosin in S. marcescens strain B2. We suggest that bacteria isolated from rice plants and rhizospheres mediate the suppression of antibiotic production of biological control agents and that such suppression is common under field conditions.     

Induced resistance to<Emphasis Type="Italic">Cladosporium cucumerinum</Emphasis> in cucumber by pectinases extracted from<Emphasis Type="Italic">Penicillium oxalicum</Emphasis>

Pectinases extract (PE) from the fermentation product ofPenicillium oxalicum BZH-2002 was tested for its ability to induce protection against scab caused byCladosporium cucumerinum on cucumber (Cucumis sativus L.) plants. Seedlings with one true leaf were sprayed with various concentrations of PE (10–200 units ml⁻¹) 3 days before inoculation withC. cucumerinum. Results showed that the induced local protection against the pathogen was dose-dependent when the concentrations of PE were between 20 and 120 units ml⁻¹; systemically induced resistance against the pathogen was not observed. Boiled PE had a slight effect on disease reduction. Commercial pectinases prepared fromAspergillus niger showed lower protection against scab compared with PE when they were used at the same concentration of enzyme activity. No inhibitory activity was observed on conidial germination or germ-tube growth ofC. cucumerinum. PE was further evaluated for its enhancement of defense-related enzymes. Peroxidase (PO), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) increased in cucumber seedlings after treatment with PE. PO and PPO remained at a higher level in PE-pretreated seedlings throughout the experiment period whether pathogen-inoculated or non-inoculated, whereas PAL activity began to decrease 2 days after PE treatment. http://www.phytoparasitica.org posting July 14, 2004.     

Suppression of rice blast using freeze-killed mycelia of biocontrol fungal candidate MKP5111B

Several applications of the suppressive fungus MKP5111B, isolated from the phylloplane of rice plants, were tested in an effort to control rice blast disease. Three treatments with MKP5111B [living (Std), killed with liquid nitrogen (FR), and autoclaved (AC)] were either sprayed onto rice seedlings or mixed into seed-sown soil. Three weeks after spraying and 4 weeks after the soil application, we introduced Magnaporthe oryzae, the causal agent of rice blast, into our systems. The Std and FR treatments suppressed rice blast, but the AC treatment proved ineffective. Although a suppressive effect was seen on new leaves, no mycelium of MKP5111B was seen. The fungus thus may have induced a systemic resistance in the rice plants. A substance from MKP5111B, such as elicitor molecule(s) are likely responsible for the induced resistance.     

茉莉酸诱导水稻幼苗对稻瘟病抗性作用研究

 外源茉莉酸处理可以显著减轻水稻幼苗稻瘟病的发生。JA诱导稻苗产生抗病性的有效浓度是0.1~0.001mmol/L,喷雾处理后2d接种稻瘟病菌获得最佳的抗病效果。0.1mmol/L JA喷雾处理后2d接种稻瘟病菌,水稻幼苗的诱抗效果都超过了55%。JA处理的诱导抗性可以维持15d左右。用0.1mmol/L JA二次强化处理水稻幼苗可以增强诱导抗病性,延长抗性的持续期。JA诱导水稻幼苗抗性机制的结果表明:0.1mmol/L JA处理可以诱导水稻体内苯丙氨酸解氨酶、过氧化物酶活性和木质素升高。     

Foliar spray of a cell wall protein fraction from the biocontrol agent <Emphasis Type="Italic">Pythium oligandrum</Emphasis> induces defence-related genes and increases resistance against Cercospora leaf spot in sugar beet

After a cell wall protein fraction (CWP) of Pythium oligandrum was sprayed on sugar beet leaves, we screened leaves for induced expression of defence-related genes and for resistance against Cercospora leaf spot. In a western blot analysis, the CWP was primarily retained on the surface of leaves without degradation for at least 48 h after spraying. In northern blot analyses, four defence-related genes (β-1, 3-glucanase, acidic class III chitinase, 5-enol-pyruvylshikimate-phosphate synthase and oxalate oxidase-like germin) were expressed more rapidly in CWP-treated leaves compared to control leaves treated with distilled water (DW). When CWP was applied to a suspension of cultured cells of sugar beet, an oxidative burst was observed that did not occur after the DW treatment. In growth chamber trials after inoculation with Cercospora beticola, the severity of Cercospora leaf spot was significantly reduced in CWP-treated plants compared to the DW-treated controls. In a field experiment, CWP treatment was also effective against the disease. CWP did not reduce growth rate of the pathogen in plate tests. The results together suggest that the CWP from P. oligandrum can be retained on the leaf surface and induce expression of disease resistance genes, thereby reducing Cercospora leaf spot on sugar beet.     

Induction of systemic resistance to <Emphasis Type="Italic">Exobasidium vexans</Emphasis> in tea through SAR elicitors

The effect of two chemical elicitors (acibenzolar-S-methyl benzo-[1,2,3]- thiadiazole-7-carboxylic acid S-methyl ester [Boost 500SC]) and salicylic acid in inducing resistance in tea plants against blister blight disease caused by Exobasidium vexans Massee, was studied. Treatments with elicitors resulted in reduced severity of blister blight disease in nursery plants on challenge with the pathogen. There was a significant increase in the activities of defense enzymes like phenylalanine ammonia lyase, peroxidase and β-1,3-glucanase on elicitor treatments in tea leaves challenged with the pathogen than on unchallenged leaves. Acibenzolar-S-methyl (ASM) at 0.14% registered the lowest disease severity (25.2%), whereas treatments with salicylic acid were inferior. Under field conditions, the application of ASM at 0.14% resulted in disease protection of 25%. When ASM was applied in alternate rounds with a standard fungicide, the disease protection improved to 46.8%. The importance of incorporating ASM as a component in integrated disease management and also its importance in organic tea cultivation is discussed.     

Biological control of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> by selected grapevine-associated bacteria and stimulation of chitinase and β-1,3 glucanase activities under field conditions

In this study, the biocontrol ability of seven grapevine-associated bacteria, previously reported as efficient against Botrytis cinerea under in vitro conditions, was evaluated in two vineyard orchards with the susceptible cv. Chardonnay during four consecutive years (2002–2005). It was shown that the severity of disease on grapevine leaves and berries was reduced to different levels, depending on the bacterial strain and inoculation method. Drenching the plant soil with these bacteria revealed a systemic resistance to B. cinerea, even without renewal of treatment. Accordingly, this resistance was associated with a stimulation of some plant defense responses such as chitinase and β-1,3-glucanase activities in both leaves and berries. In leaves, chitinase activity increased before veraison (end-July) while β-1,3-glucanase reached its maximum activity at ripening (September). Reverse patterns were observed in berries, with β-1,3-glucanase peaking at full veraison (end-August) and chitinase at a later development stage. Highest activities were observed with Acinetobacter lwoffii PTA-113 and Pseudomonas fluorescens PTA-CT2 in leaves, and with A. lwoffii PTA-113 and Pantoea agglomerans PTA-AF1 in berries. These results have demonstrated an induced protection of grapevine against B. cinerea by selected bacteria under field conditions, and suggest that induced resistance could be related to a stimulation of plant defense reactions in a successive manner.     

Using metabolic profiling to assess plant-pathogen interactions: an example using rice (<Emphasis Type="Italic">Oryza sativa)</Emphasis> and the blast pathogen <Emphasis Type="Italic">Magnaporthe grisea</Emphasis>

A metabolomics based approach has been used to study the infection of the Hwacheong rice cultivar (Oryza sativa L. cv. Hwacheong) with compatible (KJ201) and incompatible (KJ401) strains of the rice blast fungal pathogen Magnaporthe grisea. The metabolic response of the rice plants to each strain was assessed 0, 6, 12, 24, 36, and 48 h post inoculation. Nuclear Magnetic Resonance (NMR) spectroscopy and Gas and Liquid Chromatography Tandem Mass spectrometry (GC/LC-MS/MS) were used to study both aqueous and organic phase metabolites, collectively resulting in the identification of 93 compounds. Clear metabolic profiles were observed at each time point but there were no significant differences in the metabolic response elicited by each pathogen strain until 24 h post inoculation. The largest change was found to be in alanine, which was ~30% (±9%) higher in the leaves from the compatible, compared to the resistant, plants. Together with several other metabolites (malate, glutamine, proline, cinnamate and an unknown sugar) alanine exhibited a good correlation between time of fungal penetration into the leaf and the divergence of metabolite profiles in each interaction. The results indicate both that a wide range of metabolites can be identified in rice leaves and that metabolomics has potential for the study of biochemical changes in plant-pathogen interactions.     

Effect of mechanical damage on vigor,physiological parameters,and susceptibility of oat (<Emphasis Type="Italic">Avena sativa)</Emphasis> to <Emphasis Type="Italic">Fusarium culmorum</Emphasis> infection

Vigor and selected physiological parameters (content of phenolic compounds, soluble sugars, chlorophyll a and b, and carotenoids) of eight naked and two husked oat cultivars harvested at 15% moisture content were determined. Oat seeds were threshed using two rotational speeds of the threshing drum: 1.6 m s⁻¹ (LS) and 2.4 m s⁻¹ (HS). They were then inoculated with a medium pathogenicity strain of Fusarium culmorum, strain IPO 348–01. In naked cultivars, the use of HS resulted in more severe mechanical damage; in consequence, seedling vigor decreased by 16%. In naked cultivars chlorophyll a and b and carotenoids content were significantly reduced—by more than 64%—when the HS was used. The inoculation caused over a 100% increase of carbohydrates in roots at LS but only a slight increase at HS. Phenolic compound content was twice as high in roots than in leaves after inoculation for both LS and HS. Area of microdamage and reduction of root fresh weight (f.wt.) are significantly correlated with biochemical parameters. Smaller microdamage area and root f.wt. reduction are connected with higher physiological parameters, which confirms lower seedling susceptibility to pathogen infection.     

Pseudomonas syringae pv. syringae induces systemic resistance to Pyricularia oryzae in rice

Systemic resistance was induced to Pyricularia aryzae in rice by inoculation of the first leaf with the hypersensitive response causing bacterium, Pseudomonas syringae pv. syringae. Lesions caused by Pyricularia oryzae were decreased in number and size by 85% and 50%, respectively, in systemically protected leaves. Increased resistance was associated with the deposition of a dark brown material around sites of Pyricularia oryzae infection. The systemically acquired resistance was not associated with an increase in the activities of phenylalanine ammonia lyase, coniferyl alcohol dehydrogenase, peroxidase, β-1, 3-glucanase or chitinase after the challenge inocculation with Pyricularia oryzae. The levels of these enzymes were elevated by local exposure to Pseudomonas syringae pv. syringae or Pyricularia oryzae, but were not systemically induced. These data suggest that the physiological changes which occur during induced resistance in rice are different from those correlated with induced resistance in tobacco or cucumber.     

Novel methodologies in screening and selecting olive varieties and root-stocks for resistance to <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

An innovative inoculation process, involving the drilling of a trunk hole in 3 year-old olive trees and injecting a dense conidial suspension of Verticillium dahliae, was developed to study differentiation in foliar symptom expression between olive cultivars tolerant or susceptible to the pathogen. It was demonstrated that V. dahliae conidia could be translocated and colonize the xylem at the same distance above and below the point of trunk injection in both cultivars. However, the pathogen could be subsequently isolated at statistically significant percentages in susceptible cv. Amphissis compared to the tolerant cv. Kalamon, indicating operation of resistance mechanisms in the vascular phase of the disease. Consequently symptom development in the susceptible cultivar was at least sixfold more intensive compared to the tolerant cultivar, 6–11 months after trunk inoculation. Perennial olive orchard experiments, aimed at selecting Verticillium-resistant root-stocks, were conducted by applying the novel method in 2–3 year-old root-stock suckers of Amphissis olive trees and in the tolerant cvs Lianolia of Corfu and Koroneiki. It was indicated that potentially resistant root-stocks could be obtained following the trunk drilling technique. Resistance differentiation between cvs Amphissis and Kalamon was further verified through root inoculation by various V. dahliae microsclerotial concentrations and demonstrated that the trunk drilling inoculation procedure is equally efficient in resistance evaluation of olives to Verticillium wilt. The trunk inoculation procedure could be useful in selecting and screening root-stocks for resistance to V. dahliae and other vascular pathogens and could elucidate resistance mechanisms in woody plants against vascular wilt diseases.     

Characterization of <Emphasis Type="Italic">Inago1</Emphasis> and <Emphasis Type="Italic">Inago2</Emphasis> retrotransposons in <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

From the genome of a Japanese field isolate of the rice blast fungus, Magnaporthe oryzae, we newly identified Inago1 and Inago2 LTR retrotransposons. Both elements were found to be Ty3/gypsy-like elements whose copies were dispersed within the genome of Magnaporthe spp. isolates infecting rice and other monocot plants. Southern hybridization patterns of nine re-isolates derived from conidia of the strain Ina168 produced after a methyl viologen treatment were not changed, indicating that the insertion pattern of Inago elements is relatively stable.     

Post infection application of DL-3-amino-butyric acid (BABA) induces multiple forms of resistance against <Emphasis Type="Italic">Bremia lactucae</Emphasis> in lettuce

DL-3-amino-butyric acid (BABA) induces local and systemic resistance against disease in numerous plant species. In a recent study we showed that preventive application of BABA to lettuce (Lactuca sativa) plants induced resistance against downy mildew caused by the oomycete Bremia lactucae by callose encasement of the primary infection structures of the pathogen. Now we show that post-infection application of BABA to the foliage or the roots, even at progressive stages of disease development, is highly protective against B. lactucae. Resistance induced by BABA is manifested in multiple microscopic forms, depending on the time of its application. When applied at 1 day post inoculation (dpi) BABA induced HR in penetrated epidermal cells; at 2 dpi it caused massive encasement with callose of the primary haustoria; and, at 3 or 4 dpi it enhanced the accumulation of H₂O₂ in the developing mycelia runners and altered their colour to red. The pronounced change in the colour of the mycelium was visually apparent to the naked eye. In all cases the pathogen failed to sporulate on the treated plants. This is the first indication that an immunizing compound may be protective at advanced stages of disease development.     

Plant defense activation and management of tomato root rot by a chitin-fortified <Emphasis Type="Italic">Trichoderma/Hypocrea</Emphasis> formulation

Tomato root rot caused by Rhizoctonia solani is a major soilborne disease resulting in significant yield loss. The culture filtrates of six isolates of Trichoderma/Hypocrea species were evaluated for in vitro production of hydrolytic enzymes. Results demonstrated that all the six isolates were able to produce chitinase, β-1, 3 glucanase and protease in the range of 76–235 μmol GlcNAc min^-1 mg^-1 protein, 31.90–37.72 nmol glucose min^-1 mg^-1 proteins and 63.05–86.22 μmol min^-1 mg^-1 proteins, respectively. Trichoderma/Hypocrea-based formulation(s) were prepared with chitin (1% v:v) and CMC (0.5% w:v) for root rot management in a greenhouse. Root dip application with bioformulation(s) resulted in a significant reduction of the root rot index. In addition, bioformulations increased plant growth attributing traits significantly relative to untreated control. Accumulation of total phenols, peroxidase, polyphenoloxidase and phenylalanine ammonia lyase increased in chitin-supplemented Trichoderma/Hypocrea formulation-treated plants challenged with R. solani. The results suggest that chitin-fortified bioformulation(s) could be an effective system to control root rot of tomato in an eco-compatible manner.     

Comparison of root and foliar applications of potassium silicate in potentiating post‐infection defences of melon against powdery mildew

The application of silicon to the roots or leaves reduces the severity of powdery mildew (Podosphaera xanthii) in melon but the latter treatment is less effective. This study compared key biochemical defence responses of melon triggered by P. xanthii after root or foliar treatment with potassium silicate (PS). Treatments consisted of pathogen‐inoculated or mock‐inoculated plants supplied with PS via roots or foliarly, as well as a non‐treated control. The activity of defence enzymes and the concentration of phenolic compounds, lignin and malondialdehyde were determined from leaf samples at different time points after inoculation. Pathogen‐inoculated plants irrigated with PS showed both an accumulation of silicon and primed defence responses in leaves that were not observed in pathogen‐inoculated plants either sprayed with PS or not treated. These responses included the anticipated activity of peroxidase and accumulation of soluble phenols, the activation of chitinase and repression of catalase, and the stronger activation of superoxide dismutase, peroxidase and β‐1,3‐glucanase. Moreover, the lignin concentration increased in response to inoculation, whereas the malondialdehyde concentration decreased. For the foliar treatment, however, only an increase in lignin deposition was observed compared with the control plants. The results show that silicon strongly plays an active role in modulating the defence responses of melon against P. xanthii when supplied to the roots as opposed to the foliage.     

Site‐directed mutagenesis of the cytochrome b gene and development of diagnostic methods for identifying QoI resistance of rice blast fungus

BACKGROUND: It is possible that a single nucleotide polymorphism (SNP) (G143A mutation) in the cytochrome b gene could confer resistance to quinone outside inhibiting (QoI) fungicides (strobilurins) in rice blast fungus because this mutation caused a high level of resistance to fungicides such as azoxystrobin in Pyricularia grisea Sacc. and other fungal plant pathogens. The aim of this study was to survey Magnaporthe oryzae B Couch sp. nov. isolates in Japan for resistance to QoIs, and to try to develop molecular detection methods for QoI resistance. RESULTS: A survey on the QoI resistance among M. oryzae isolates from rice was conducted in Japan. A total of 813 single‐spore isolates of M. oryzae were tested for their sensitivity to azoxystrobin using a mycelial growth test on PDA. QoI fungicide resistance was not found among these isolates. The introduction of G143A mutation into a plasmid containing the cytochrome b gene sequence of rice blast fungus was achieved by site‐directed mutagenesis. Molecular diagnostic methods were developed for identifying QoI resistance in rice blast fungus using the plasmid construct. CONCLUSION: As the management of rice blast disease is often dependent on chemicals, the rational design of control programmes requires a proper understanding of the fungicide resistance phenomenon in field populations of the pathogen. Mutation of the cytochrome b gene of rice blast fungus would be specifically detected from diseased leaves and seeds using the molecular methods developed in this study. Copyright © 2009 Society of Chemical Industry     

Green-odour compounds have antifungal activity against the rice blast fungus <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Four green-odour compounds—trans-2-hexenal, cis-3-hexenol, n-hexanal, and cis-3-hexenal—were applied (0.85 μg ml⁻¹ as vapour) to rice plants in laboratory conditions to observe their biological activity against the phytopathogenic fungus Maganporthe oryzae, which causes rice blast disease worldwide. Two compounds, trans-2-hexenal and cis-3-hexenal, showed remarkable disease suppression efficacy (99.7% and 100% suppression, respectively), while n-hexanal had moderate (86.5%) and cis-3-hexenol had weak (20.8%) disease-suppressing effects. Pre-application and post-application of trans-2-hexenal or cis-3-hexenal had slight effects on blast incidence, suggesting that these compounds had direct effects to suppress M. oryzae infection. In fact, trans-2-hexenal and cis-3-hexenal exhibited a growth suppression effect on M. oryzae. Interestingly, these two compounds inhibited appressorium formation at lower concentrations than the growth suppression. Studies on the hypersensitive response (HR)-like reaction and plant β-1,3-glucanase activity in rice plant confirmed that induced resistance was not the major factor involved in the disease suppression mechanism. Results of this study conclusively showed that trans-2-hexenal and cis-3-hexenal possess potent inhibitory activities against the growth and the appressorium formation of M. oryzae and could be used as antifungal agents to significantly reduce M. oryzae infections in rice.     

Induced Resistance as a Mechanism of Biological Control by Lysobacter enzymogenes Strain C3

ABSTRACT Induced resistance was found to be a mechanism for biological control of leaf spot, caused by Bipolaris sorokiniana, in tall fescue (Festuca arundinacea) using the bacterium Lysobacter enzymogenes strain C3. Resistance elicited by C3 suppressed germination of B. sorokiniana conidia on the phylloplane in addition to reducing the severity of leaf spot. The pathogen-inhibitory effect could be separated from antibiosis by using heat-inactivated cells of C3 that retained no antifungal activity. Application of live or heat-killed cells to tall fescue leaves resulted only in localized resistance confined to the treated leaf, whereas treatment of roots resulted in systemic resistance expressed in the foliage. The effects of foliar and root applications of C3 were long lasting, as evidenced by suppression of conidial germination and leaf spot development even when pathogen inoculation was delayed 15 days after bacterial treatment. When C3 population levels and germination of pathogen conidia was examined on leaf segments, germination percentage was reduced on all segments from C3-treated leaves compared with segments from non-treated leaves, but no dose-response relationship typical of antagonism was found. Induced resistance by C3 was not host or pathogen specific; foliar application of heat-killed C3 cells controlled B. sorokiniana on wheat and also was effective in reducing the severity of brown patch, caused by Rhizoctonia solani, on tall fescue. Treatments of tall fescue foliage or roots with C3 resulted in significantly elevated peroxidase activity compared with the control.     

Silicon-enhanced resistance to rice blast is attributed to silicon-mediated defence resistance and its role as physical barrier

A series of experiments were performed to study the effects of silicon (Si) on rice blast development, H₂O₂ accumulation and lipid peroxidation in a controlled rice—Magnaporthe grisea pathosystem. Rice plants supplied with Si as a single dose immediately after pathogen inoculation (−/+Si) exhibited the same high protection against disease as plants treated continuously with Si for the whole growth period (+/+Si), with disease severity indices of 20.8% and 19.6%, respectively, which were significantly lower than that for the control treatment with no Si supplied (63.7%). A single application of Si to rice plants before inoculation (+/−Si) conferred partial protection (disease severity index of 33.3%) compared with the control treatment. Silicon induced a rapid but transient burst of H₂O₂ at 24 h after inoculation. The addition of Si to rice plants significantly altered the activities of catalase and lipoxygenase and the concentration of malodialdehyde (indicative of lipid peroxidation) in rice plants. We propose that rice plants may respond to Si by increased H₂O₂ accumulation and lipid peroxidation. In turn, these responses are linked to host defence mechanisms such as lignin production, oxidative cross-linking in the cell wall, phytoalexin production, and the hypersensitive reaction. Thus, the mechanisms of Si-stimulated plant disease protection may extend beyond its established role in physically strengthening cell walls.     

Biological control of rice blast by the epiphytic bacterium <Emphasis Type="Italic">Erwinia ananas</Emphasis> transformed with a chitinolytic enzyme gene from an antagonistic bacterium, <Emphasis Type="Italic">Serratia marcescens</Emphasis> strain B2

The gene chiA, encoding for the endochitinase ChiA, was cloned from Serratia marcescens strain B2, a tomato epiphytic bacterium, and introduced into the epiphytic bacterium Erwinia ananas NR-1, isolated from rice phylloplane. The gene chiA was introduced under the control of two types of promoter into a broad-host-range plasmid vector. The vector contained various fragments with promoter activity isolated from E. ananas chromosomal DNA. The constructed vectors were designated pchiA-V1pcf9 and pchiA-V1pcf53 for their respective promoters. E. ananas NR-1 transformed with either of these vectors produced and secreted ChiA. The antifungal activity of ChiA produced by transformed E. ananas NR-1 was demonstrated in vitro by the inhibition of Pyricularia oryzae germ tube elongation such as bursting of the hyphal tip. Transformed E. ananas NR-1 suppressed the incidence of rice blast caused by P. oryzae under greenhouse conditions; however, the magnitude of the suppressive effect depended on which promoter was used. Both transformants and the nontransformant E. ananas NR-1 survived on rice phylloplane. It is expected that the rice epiphytic bacterium E. ananas NR-1 carrying a chitinolytic enzyme gene is an efficient biological control agent against rice blast.