Antimicrobial Activities of Selected Cyathus Species

Twelve selected Cyathus species were tested for their abilities to produce antimicrobial metabolites. Most of them were found to produce secondary exo-metabolites that could induce morphological abnormalities of rice pathogenic fungi Pyricularia oryzae. Some extracts from the cultivated liquid obviously inhibited human pathogenic fungi Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans. Activities against six human pathogenic bacteria were also obtained from some of these extracts.     

Control of blast and sheath blight diseases of rice using antifungal metabolites produced by Streptomyces sp. PM5

Two antifungal aliphatic compounds, SPM5C-1 and SPM5C-2 with a lactone and ketone carbonyl unit, respectively obtained from Streptomyces sp. PM5 were evaluated under in vitro and in vivo conditions against major rice pathogens, Pyricularia oryzae and Rhizoctonia solani. These compounds were dissolved in distilled water/medium to get the required concentrations. The well diffusion bioassay indicated that the of SPM5C-1 remarkably inhibited the mycelial growth of P. oryzae and R. solani in comparison to SPM5C-2. Though SPM5C-2 showed low antifungal activity against P. oryzae, it was not active against R. solani. Further, SPM5C-1 completely inhibited the growth of P. oryzae and R. solani at concentrations of 25, 50, 75 and 100 μg/ml. Greenhouse experiments revealed that spraying of SPM5C-1 at 500 μg/ml on rice significantly decreased blast and sheath blight development by 76.1% and 82.3%, respectively, as compared to the control with a corresponding increase in rice grain yield.     

Pseudomonas aeruginosa inducing rice resistance againstRhizoctonia solani: Production of salicylic acid and peroxidases

Three isolates of Pseudomonas aeruginosa were used for seed treatment of rice; all showed plant growth promoting activity and induced systemic resistance in rice against Rhizoctonia solani G5 and increased seed yield. Production of salicylic acid (Sal) by P. aeruginosa both in vitro and in vivo was quantified with high performance liquid chromatography. All three isolates produced more Sal in King's B broth than in induced roots. Using a split root system, more Sal accumulated in root tissues of bacterized site than in distant roots on the opposite site of the root system after 1 d, but this difference decreased after 3 d. Sal concentration 0-200 g/L showed no inhibition of mycelial growth of R. solani in vitro, while at > or =300 g/L it inhibited it. P. aeruginosa-pretreated rice plants challenged inoculation with R. solani (as pathogen), an additional increase in the accumulation of peroxidase was observed. Three pathogenesis-related peroxidases in induced rice plants were detected; molar mass of these purified peroxidases was 28, 36 and 47 kDa. Purified peroxidase showed antifungal activity against phytopathogenic fungi R. solani, Pyricularia oryzae and Helminthosporium oryzae.     

Three types of defense-responsive genes are involved in resistance to bacterial blight and fungal blast diseases in rice

Bacterial blight and fungal blast diseases of rice, caused by Xanthomonas oryzae pv. oryzae and Pyricularia grisea Sacc., respectively, are two of the most devastating diseases in rice worldwide. To study the defense responses to infection with each of these pathogens, expression profiling of 12 defense-responsive genes was performed using near-isogenic rice lines that are resistant or susceptible to bacterial blight and fungal blast, respectively, and rice cultivars that are resistant or susceptible to both pathogens. All 12 genes showed constitutive expression, but expression levels increased in response to infection. Based on their expression patterns in 12 host-pathogen combinations, these genes could be classified into three types, pathogen non-specific (6), pathogen specific but race non-specific (4) and race specific (2). Most of the 12 genes were only responsive during incompatible interactions. These results suggest that bacterial blight and fungal blast resistances share common pathway(s), but are also regulated by different defense pathways in rice. Activation of the corresponding R gene is the key step that initiates the action of these genes in defense responses. The chromosomal locations and pathogen specificities of seven of the 12 genes were consistent with those of previously identified quantitative trait loci for rice disease resistance, which indicates that some of the 12 genes studied may have a phenotypic impact on disease resistance in rice.     

Plant--fungal interactions: the search for phytoalexins and other antifungal compounds from higher plants

A brief review is given of some biological, chemical and chemotaxonomic aspects of phytoalexin research. Emphasis is placed on the search for antifungal compounds in the plant families Leguminosae and Rosaceae, and in rice, Oryza sativa. The possible role of phytoalexins in the resistance of rice plants against the fungus Pyricularia oryzae (= Magnaporthe grisea) is discussed, and the future prospects of phytoalexin research are outlined.     

Biological control of rice bacterial blight by plant-associated bacteria producing 2,4-diacetylphloroglucinol

Certain plant-associated strains of fluorescent Pseudomonas spp. are known to produce the antimicrobial antibiotic 2,4-diacetylphloroglucinol (DAPG). It has antibacterial, antifungal, antiviral, and antihelminthic properties and has played a significant role in the biological control of tobacco, wheat, and sugar beet diseases. It has never been reported from India and has not been implicated in the biological suppression of a major disease of the rice crop. Here, we report that a subpopulation of 27 strains of plant-associated Pseudomonas fluorescens screened in a batch of 278 strains of fluorescent pseudomonads produced DAPG. The DAPG production was detected by a PCR-based screening method that used primers Phl2a and Phl2b and amplified a 745-bp fragment characteristic of DAPG. HPLC, 1H NMR, and IR analyses provided further evidence for its production. We report also that this compound inhibited the growth of the devastating rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae in laboratory assays and suppressed rice bacterial blight up to 59%-64% in net-house and field experiments. Tn5 mutants defective in DAPG production (Phl-) of P. fluorescens PTB 9 were much less effective in their suppression of rice bacterial blight.     

A potato carboxypeptidase inhibitor gene provides pathogen resistance in transgenic rice

A defensive role against insect attack has been traditionally attributed to plant protease inhibitors. Here, evidence is described of the potential of a plant protease inhibitor, the potato carboxypeptidase inhibitor (PCI), to provide resistance to fungal pathogens when expressed in rice as a heterologous protein. It is shown that rice plants constitutively expressing the pci gene exhibit resistance against the economically important pathogens Magnaporthe oryzae and Fusarium verticillioides . A M. oryzae carboxypeptidase was purified by affinity chromatography and further characterized by mass spectrometry. This fungal carboxypeptidase was found to be a novel carboxypeptidase B which was fully inhibited by PCI. Overall, the results indicate that PCI exerts its antifungal activity through the inhibition of this particular fungal carboxypeptidase B. Although pci confers protection against fungal pathogens in transgenic rice, a significant cost in insect resistance is observed. Thus, the weight gain of larvae of the specialist insect Chilo suppressalis (striped stem borer) and the polyphagous insect Spodoptera littoralis (Egyptian cotton worm) fed on pci rice is significantly larger than that of insects fed on wild-type plants. Homology-based modelling revealed structural similarities between the predicted structure of the M. oryzae carboxypeptidase B and the crystal structure of insect carboxypeptidases, indicating that PCI may function not only as an inhibitor of fungal carboxypeptidases, but also as an inhibitor of insect carboxypeptidases. The potential impact of the pci gene in terms of protection against fungal and insect diseases is discussed.     

Transgenic indica Rice Expressing ns-LTP-Like Protein Shows Enhanced Resistance to Both Fungal and Bacterial Pathogens

Antimicrobial peptides (AMPs) from plant seeds, known to inhibit pathogen growth have a great potential in developing transgenic plants resistant to disease. Some of the nonspecific-lipid transfer proteins (ns-LTP) that facilitate in vitro transport of lipids, show antimicrobial activity in vitro. Rice seeds also contain ns-LTPs; however, these genes are expressed weakly in seedlings. We have transformed Pusa Basmati 1, an elite indica rice cultivar, with the gene for Ace-AMP1 from Allium cepa, coding for an effective antimicrobial protein homologous to ns-LTPs. The gene for Ace-AMP1 was cloned under an inducible rice phenylalanine ammonia-lyase (PAL) or a constitutive maize ubiquitin (UbI) promoter. Ace-AMP1 was expressed in transgenic lines and secreted in the apoplastic space. Protein extracts from leaves of transgenic plants inhibited three major rice pathogens, Magnaporthe grisea, Rhizoctonia solani and Xanthomonas oryzae, in vitro. Enhanced resistance against these pathogens was observed in in planta assays, and the degree of resistance correlating with the levels of Ace-AMP1 with an average increase in resistance to blast, sheath blight, and bacterial leaf blight disease by 86%, 67%, and 82%, respectively. Importantly, transgenic rice plants, with stable integration and expression of Ace-AMP1, retained their agronomic characteristics while displaying enhanced resistance to both fungal and bacterial pathogens.     

The defense-responsive genes showing enhanced and repressed expression after pathogen infection in rice (Oryza sativa L.)

Despite large numbers of studies about defense response, processes involved in the resistance of plants to incompatible pathogens are still largely uncharacterized. The objective of this study was to identify genes involved in defense response by cDNA array analysis and to gain knowledge about the functions of the genes involved in defense response. Approximately 20000 rice cDNA clones were arrayed on nylon filters. RNA samples isolated from different rice lines after infection with incompatible strains or isolates of Xanthomonas oryzae pv. oryzae or Pyricularia grisea, respectively, were used to synthesize cDNA as probes for screening the cDNA arrays. A total of 100 differentially expressed unique sequences were identified from 5 pathogen-host combinations. Fifty-three sequences were detected as showing enhanced expression and 47 sequences were detected as showing repressed expression after pathogen infection. Sequence analysis revealed that most of the 100 sequences had various degrees of homology with     

植物抗毒素转化水稻和转基因植株的生物鉴定

用基因枪法转化了水稻（ＯｒｙｚａｓａｔｉｖａＬ．）６个材料的未成熟胚、成熟胚及胚性愈伤组织。质粒ｐＳＳＶｓｔ１和ｐＶＥ５＋是由葡萄中分离出的编码芪类合成酶的植物抗毒素基因与３５Ｓ或它自己的启动子组成。Ｇ４１８（１００～１５０ｍｇ／Ｌ）或潮霉素（５０ｍｇ／Ｌ）筛选后,经ＰＣＲ、Ｓｏｕｔｈｅｒｎｂｌｏｔ或Ｄｏｔｂｌｏｔ分析证明的转基因植株共５４株。对转基因植株及其后代进行了稻瘟病和白叶枯病的抗性鉴定。初步结果表明,芪类合成酶基因可以提高转基因植株及后代的抗性。     

Screening of Antifungal Protein Producing Strain TG26 of Bacillus spp. and Studies on Its Cultrual Conditions

An antagonistic strain, Bacillus spp. TG26 isolated from roots of a towel gourd (Luffa cylindrica (L.) Roem.), secretes large amount of antifungal proteins with strong inhibiting activity against Gibberella zeae, Fusarium oxysporum f. vasinfectum and niveum, ,4hernarict longipes, Trichoderma viride as well as bacterial pathogens of Xanthomonas campestris pv. oryzae and Psendomonas solanaceanum. Crude proteins obtained by precipitation of the cellfree culture of TG26 strain with ammonium sulphate at 70% saturation are over 130 mg/L. The optimal culture conditions of TG26 strain are as following: BPY medium, initial pH 7.0, temperature 30 ℃ and cultivation time 36 hours.     

水稻对白叶枯病和细菌性条斑病的抗性遗传研究进展

水稻白叶枯病和水稻细菌性条斑病是由稻黄单胞细菌(Xanthomonas oryzae)不同致病变种引起的两种最重要的水稻细菌性病害。发掘和利用抗性基因,培育抗病品种是防治这两种病害的最有效手段之一。本文分别综述了这两种高度相关的病害的抗性遗传研究进展,包括已发掘和利用的主效抗性基因特点及目前国内外对这两种病害的抗性QTL定位研究进展,为水稻抗白叶枯病和细菌性条斑病育种研究提供有用信息。     

Rice Transformation with a Phytoalexin Gene and Bioassay of the Transgenic Plants

Immature embryos, mature embryos and embryogenie ealli of 6 rice ( Oryza sativa L. ) materials were transformed with particle bombardment. The plasmids pSSVsfl and pVE5 + were used, both containing the phytoalexin gene from grapevine coding for stilbene synthase, but driven by 35S and its own promoter respectively. Through resistance selection for G418 ( 100 to 150 mg/L) or hygromycin (50 mg/L), 54 independent transgenic plants were isolated and further assessed by PCR, Southern blot and Dot blot analyses. The transgenic plants and their progenies were tested for resistance to blast ( Pyricularia oryzae) and bacterial blight of rice ( Xanthomonas oryzae). Preliminary results indicated that the stilbene synthase gene could enhance the resistance of transgenic plants and their progenies to both pathogens.     

Functional interplay between two Xanthomonas oryzae pv,. oryzae secretion systems in modulating virulence on rice

The type II (T2S) and type III (T3S) secretion systems are important for virulence of Xanthomonas oryzae pv. oryzae, causal agent of bacterial leaf blight of rice. The T3S of gram-negative bacterial plant pathogens has been shown to suppress host defense responses, including programmed cell death reactions, whereas the T2S is involved in secreting cell-wall-degrading enzymes. Here, we show that a T3S-deficient (T3S-) mutant of X. oryzae pv. oryzae can induce a basal plant defense response seen as callose deposition, immunize rice against subsequent X. oryzae pv. oryzae infection, and cause cell-death-associated nuclear fragmentation. A T2S- T3S- double mutant exhibited a substantial reduction in the ability to evoke these responses. We purified two major effectors of the X. oryzae pv. oryzae T2S and characterized them to be a cellulase (ClsA) and a putative cellobiosidase (CbsA). The purified ClsA, CbsA, and lipase/esterase (LipA; a previously identified T2S effector) proteins induced rice defense responses that were suppressible by X. oryzae pv. oryzae in a T3S-dependent manner. These defense responses also were inducible by the products of the action of these purified proteins on rice cell walls. We further show that a CbsA- mutant or a ClsA- LipA- double mutant are severely virulence deficient. These results indicate that the X. oryzae pv. oryzae T2S secretes important virulence factors, which induce innate rice defense responses that are suppressed by T3S effectors to enable successful infection.     

Recent Advances in Cloning and Characterization of Disease Resistance Genes in Rice

Rice diseases caused by fungi, bacteria and viruses are one of the major constraints for sustainable rice （Oryza sativa L.） production worldwide. The use of resistant cultivars is considered the most economical and effective method to control rice diseases. In the last decade, a dozen resistance genes against the fungal pathogen Magnaporthe grisea and the bacterial pathogen Xanthomonas oryzae pv. oryzae have been cloned. Approximately half of them encode nuclear binding site （NBS） and leucine rich repeat （LRR）-containing proteins, the most common type of cloned plant resistance genes. Interestingly, four of them encode novel proteins which have not been identified in other plant species, suggesting that unique mechanisms might be involved in rice defense responses. This review summarizes the recent advances in cloning and characterization of disease resistance genes in rice and presents future perspectives for in-depth molecular analysis of the function and evolution of rice resistance genes and their interaction with avirulence genes in pathogens.     

A protein from the mold Aspergillus giganteus is a potent inhibitor of fungal plant pathogens.

A purified preparation of antifungal protein (AFP) from Aspergillus giganteus exhibited potent antifungal activity against the phytopathogenic fungi Magnaporthe grisea and Fusarium moniliforme, as well as the oomycete pathogen Phytophthora infestans. Under conditions of total inhibition of fungal growth, no toxicity of AFP toward rice protoplasts was observed. Additionally, application of AFP on rice plants completely inhibited M. grisea growth. These results are discussed in relation to the potential of the afp gene to enhance crop protection against fungal pathogens in transgenic plants.     

Manipulating broad-spectrum disease resistance by suppressing pathogen-induced auxin accumulation in rice

Breeding crops with the quality of broad-spectrum disease resistance using genetic resources is one of the principal goals of crop improvement. However, the molecular mechanism of broad-spectrum resistance remains largely unknown. Here, we show that GH3-2, encoding an indole-3-acetic acid (IAA)-amido synthetase, mediates a broad-spectrum resistance to bacterial Xanthomonas oryzae pv oryzae and Xanthomonas oryzae pv oryzicola and fungal Magnaporthe grisea in rice (Oryza sativa). IAA, the major form of auxin in rice, results in rice more vulnerable to the invasion of different types of pathogens, which is at least partly due to IAA-induced loosening of the cell wall, the natural protective barrier of plant cells to invaders. X. oryzae pv oryzae, X. oryzae pv oryzicola, and M. grisea secrete IAA, which, in turn, may induce rice to synthesize its own IAA at the infection site. IAA induces the production of expansins, the cell wall-loosening proteins, and makes rice vulnerable to pathogens. GH3-2 is likely contributing to a minor quantitative trait locus for broad-spectrum resistance. Activation of GH3-2 inactivates IAA by catalyzing the formation of an IAA-amino acid conjugate, which results in the suppression of expansin genes. Thus, GH3-2 mediates basal resistance by suppressing pathogen-induced IAA accumulation. It is expected that, regulated by a pathogen-induced strong promoter, GH3-2 alone may be used for breeding rice with a broad-spectrum disease resistance.     

Two compounds from allelopathic rice accession and their inhibitory activity on weeds and fungal pathogens

A flavone (5,7,4'-trihydroxy-3',5'-dimethoxyflavone), a cyclohexenone (3-isopropyl-5-acetoxycyclohexene-2-one-1) and a liquid mixture of low polarity, containing long-chain and cyclic hydrocarbons, were isolated from leaves of allelopathic rice accession PI 312777 using column chromatography. Their structures and constituents were identified by means of HR-MS, NMR and GC/MS analyses, respectively. Bioassays showed that both the flavone and cyclohexenone significantly inhibited the growth of weeds Echinochloa crus-galli, Cyperus difformis and Cyperus iris, and the spore germination of fungal pathogens Pyricularia oryzae and Rhizoctonia solani at all tested concentrations. Moreover, the combination of the inactive mixture of low polarity and the active flavone or cyclohexenone significantly enhanced the inhibitory activities on weed growth. In addition, the two compounds and the mixture of low polarity from the leaves of PI312777 did not inhibit the rice growth at the same concentrations. It was also established that both compounds could be released into the soil, and was especially induced by E. crus-galli. The results suggest that 5,7,4'-trihydroxy-3',5'-dimethoxyflavone and 3-isopropyl- 5-acetoxycyclohexene-2-one-1 may act as allelochemicals participating in the defense of rice against weeds and pathogens.     

Synthesis and fungicidal activity of new N,O-acyl chitosan derivatives

Novel N,O-acyl chitosan (NOAC) derivatives were synthesized to examine their fungicidal activity against the gray mould fungus Botrytis cinerea (Leotiales: Sclerotiniaceae) and the rice leaf blast fungus Pyricularia oryzae (Teleomorph: Magnaporth grisea). The fungicidal activity was evaluated by the radial growth bioassay. NOAC derivatives were more active against the two plant pathogens than chitosan itself, and the effect was concentration dependent. Against B. cinerea, 4-chlorobutyryl chitosan (EC50=0.043%), decanoyl chitosan (EC50=0.044%), cinnamoyl chitosan (EC50=0.045%), and p-methoxybenzoyl chitosan (EC50=0.050%) were the most active (12-13-fold more active than chitosan). (Un)-substituted benzoyl chitosan derivatives were more active against B. cinerea than most of these with N,O-alkyl derivatives. Against P. oryzae chitosan derivatives with lauroyl, methoxy acetyl, methacryloyl and decanoyl were the most active.     

Exopolysaccharides of Xanthomonas pathovar strains that infect rice and wheat crops

In order to understand the mode of action of the taxonomically related pathogens Xanthomonas campestris pv. translucens, Xanthomonas oryzae pv. oryzae, and Xanthomonas oryzae pv. oryzicola, which attack wheat and rice crops, we examined the compositional differences of their exopolysaccharides (EPSs). Maximum production of polysaccharide in shake cultures of these pathogens was observed between 24 and 72 h. X. campestris pv. translucens, the leaf streak pathogen of wheat, produced a higher amount of polysaccharide (46.97 microg/ml) at 72 h compared to X. oryzae pv. oryzae (42.02 microg/ml), the bacterial blight pathogen of rice, and X. oryzae pv. oryzicola (41.91 microg/ml), the bacterial leaf streak pathogen of rice. Infrared (FTIR) spectra suggested that the polysaccharides of all three Xanthomonas pathovar strains have an -OH group with intermolecular hydrogen bonding, a C-H group of methyl alkanes, an aldehyde (RCHO) group, a C=C or C=O group, and a C-O group. FTIR spectra also revealed the presence of an acid anhydride group in X. oryzae pv. oryzae, a secondary aromatic or aliphatic amine group in X. campestris pv. translucens, and a primary aromatic or aliphatic amine group in X. oryzae pv. oryzae and X. oryzae pv. oryzicola. Nuclear magnetic resonance (NMR) spectra revealed the presence of unsubstituted sugars, an acetyl amine of hexose or pentose, and a beta-anomeric carbon of hexose or pentose in the polysaccharides of all bacteria. NMR spectra also identified the alpha-anomeric carbon of hexose or pentose in all strains, and a branching at the fourth carbon of the sugar only in X. campestris pv. translucens; the presence of an uronic acid molecule (acid anhydride group) in X. oryzae pv. oryzae; and a deoxy sugar, rhamnose, in X. oryzae pv. oryzicola.