Differential susceptibility ofphialophora gregata ff.sp.adzukicola andsojae to antimicrobial chemicals

The susceptibility ofPhialophora gregata ff.sp.adzukicola andsojae to antimicrobial chemicals was investigated. The minimum inhibitory concentrations (MICs) of benomyl, chloramphenicol, CuSO₄, cycloheximide and perchlorate for mycelial growth were the same for the two formae speciales. The MIC of hygromycin against f.sp.adzukicola was slightly lower than that against f.sp.sojae, and the latter was more resistant to iprodion than the former. Susceptibility to nystatin was markedly different: ff.sp.adzukicola andsojae had relative growth values of 3–20% and 59–93% at 100 µg/ml, respectively, and this difference could be used to differentiate the two formae speciales.     

Characterization of a group I intron in the nuclera rDNA differentiatingPhialophora gregata f. sp.adzukicola fromP. gregata f. sp.sojae

An insertion sequence was detected near the 3′ end of the nuclear small subunit rDNA in isolates ofPhialophora gregata f. sp.adzukicola, the causal agent of the brown stem rot disease of adzuki bean. This insertion sequence was absent in isolates ofP. gregata, f. sp.sojae which causes brown stem rot of soybean. The insertion sequence is 304 bp long and contains all the characteristics of group I introns. These characteristics include, the four conserved sequence elements (P, Q, R, and S), a U at the 5′ splice site of the exon, a G at the 3′ splice site of the intron, a putative internal guiding sequences; the sequence also fits a secondary structure model for group I introns. Similar to most group I introns found in nuclear small subunit rDNA, the intron was located in a highly conserved region and is devoid of long open reading frames. This intron provides a convenient marker for use in conventional PCR to separateP. gregata f. sp.adzukicola fromP. gregata f. sp.sojae.     

大豆根瘤内抗棉花枯萎病菌株的筛选及其防病试验

【目的】采用优良抗病性内生菌资源来控制棉花枯萎病是一种有效的措施。本研究从大豆根瘤中筛选棉花枯萎病拮抗性内生细菌,探索其对棉花枯萎病菌丝的抑制作用和代表菌株特性,为发掘和应用防病、抗逆优良菌株提供理论基础。【方法】采用对峙法和代谢液培养法对大豆根瘤内生细菌进行棉花枯萎病菌抑菌性筛选,显微观察法研究筛选菌株引起病原菌菌丝变化,通过菌株培养特征、理化特性和16S r DNA序列同源性分析确定菌株系统发育地位,比色法测定DD174耐盐碱性,盆栽试验验证防病效果。【结果】经复筛和代谢液试验有5株拮抗性较强菌株,被作用病原菌菌丝畸形、细胞壁消失、自溶,菌丝基部加粗、分支增多,呈树根状;菌丝被菌苔包埋而溶解、断裂,菌丝末端球形膨大。对棉花枯萎病菌的抑制作用主要通过菌体产生胞外代谢物发挥作用。菌株DD174、DD176和DD179最相似菌株分别为Bacillus oceanisediminis H2T(GQ292772)和B.thuringiensis ATCC 10792T(AF290545),菌株DD165和DD166最相似菌株均为Stenotrophomonas maltophilia LMG 958T(X95923)。DD174能耐受6%盐浓度,p H 10生长良好,具有一定耐盐碱能力。DD174处理组防治效果达76.32%,其他防效均在62%以上,可作为棉花枯萎病的生防菌株资源。【结论】大豆根瘤内存在棉花枯萎病内生拮抗细菌,其中有些菌株具有一定耐盐碱能力,对棉花枯萎病病原菌及病害有一定抑菌和防病作用。     

Induced chitinase activity in resistant wheat leaves inoculated with an incompatible race of Puccinia striiformis f. sp. tritici,the causal agent of yellow rust disease

Chitinase specific activity was measured spectrophotometrically in wheat leaf tissues during the compatible and incompatible interactions with Puccinia striiformis f. sp. tritici, the causal agent of yellow rust disease. The wheat cultivar, Federation^* 4/Kavkaz, was inoculated with virulent (134E134A+) or avirulent (4EOA+) races of P. striiformis f. sp. tritici in the first leaf stage. The results showed that chitinase activity pattern was similar in both compatible and incompatible interactions up to 72 hrs after inoculation. However, the specific activity increased rapidly in the incompatible reaction thereafter. In susceptible reaction, chitinase activity gradually declined after 72 hrs post-inoculation reaching a level similar to that in the control plants two weeks after inoculation. Chitinase specific activity in resistance response was at least three times greater than that in the susceptible reaction two weeks following the inoculation. Electrophoresis of native polyacrylamide gel impregnated with 0.1% (w/v) glycol chitinas the substrate revealed the presence of eight chitinase isoforms with relative electrophoretic mobility (R_m) values ranging from 0.11 to 0.64 in the resolving gel. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evaluation of Streptomyces sp. strain g10 for suppression of Fusarium wilt and rhizosphere colonization in pot-grown banana plantlets

Streptomyces sp. strain g10 exhibited strong antagonism towards Fusarium oxysporum f.sp. cubense (Foc) races 1, 2 and 4 in plate assays by producing extracellular antifungal metabolites. Treating the planting hole and roots of 4-week-old tissue-culture-derived Novaria banana plantlets with strain g10 suspension (10⁸ cfu/ml), significantly (P<0.05) reduced wilt severity when the plantlets were inoculated with 10⁴ spores/ml Foc race 4. The final disease severity index for leaf symptom (LSI) and rhizome discoloration (RDI) was reduced about 47 and 53%, respectively, in strain g10-treated plantlets compared to untreated plantlets. Reduction in disease incidence was not significant (P<0.05) when plantlets were inoculated with a higher concentration (10⁶ spores/ml) of Foc race 4. Rhizosphere population of strain g10 showed significant (P=0.05) increase of more than 2-fold at the end of the 3rd week compared to the 2nd week after soil amendment with the antagonist. Although the level dropped, the rhizosphere population at the end of the 6th week was still nearly 2-fold higher than the level detected after 2 weeks. In contrast, the root-free population declined significantly (P=0.05), nearly 4-fold after 6 weeks when compared to the level detected after 2 weeks. Neither growth-inhibiting nor growth-stimulating effects were observed in plantlets grown in strain g10-amended soil.     

Management Fusarium wilt on melon and watermelon by Penicillium oxalicum

The potential of the biological control fungus Penicillium oxalicum to suppress wilt caused by Fusarium oxysporum f. sp. melonis and F. oxysporum f. sp. niveum on melon and watermelon, respectively, was tested under different growth conditions. The area under disease progress curve of F. oxysporum f. sp. melonis infected melon plants was significantly reduced in growth chamber and field experiments. In glasshouse experiments, it was necessary to apply P. oxalicum and dazomet in order to reduce Fusarium wilt severity in melons caused by F. oxysporum f. sp. melonis. For watermelons, we found that P. oxalicum alone reduced the area under the disease progress curve by 58% in the growth chamber experiments and 54% in the glasshouse experiments. From these results, we suggested that P. oxalicum may be effective for the management of Fusarium wilt in melon and watermelon plants.     

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.     

The determination of physiological race of Fusarium oxysporum f. sp. lycopersici of tomato in Zhejiang, China

A group of differential tomato lines was used to identify the races of Fusarium oxysporum f. sp. lycopersici in Zhejiang, China. Marmande verte carries no resistant genes and Marporum carries gene I-1. Both lines Motelle and Mogeor have Gene I-1 and I-2. Tomato seedlings of eighteen days after sowing were inoculated with an isolate of Fusarium oxysporum f. sp. lycopersici, No. 98-2 and kept in a growth chamber. The seedlings were evaluated at fourteen days after inoculation. Results showed that Marmande verte and Marporum were severely infected by the pathogen and established as susceptible. Motelle and Mogeor were not infected and established as resistant. These results indicated that the isolate No. 98-2 represented the race 2 of Fusarium oxysporum f. sp. lycopersici and gene I-2 is necessary for obtaining resistance to this pathogen in the Zhejiang region.     

Effect of two microalgae concentrations on body size and egg size of the rotifer <Emphasis Type="Italic">Brachionus calyciflorus</Emphasis>

The rotifer, Brachionus calyciflorus, was grown with two algae species (Chlorella sp. and Scenedesmus obliquus) at different concentrations (0.1, 1 and 10 × 10⁶ cells ml⁻¹). The body size (lorica biovolume) of individual rotifer and their egg size were measured when the populations were roughly in the exponential phase of population growth. The body size of the rotifers differed significantly (P < 0.05) among the two algae species used, however this effect was not observed for egg size. The body size of rotifers fed on higher densities of Chlorella sp. (10 × 10⁶ cells ml⁻¹) was significantly larger than for those fed on lower and medium densities (0.1 and 1 × 10⁶ cells ml⁻¹). Body size and egg size of rotifers fed with different amounts of Scenedesmus did not differ significantly. The egg size was significantly larger at higher food level of Chlorella. A significantly positive correlation was observed between the adult rotifer body size and their egg size.     

Modification of competence for in vitro response to Fusarium oxysporum in tomato cells. I. Selection from a susceptible cultivar for high and low polysaccharide content

Plant cell walls play a major role in the outcome of host-parasite interactions. Wall fragments released from the plant, and/or the fungal pathogen, can act respectively as endogenous and exogenous elicitors of the defence response, and other wall components, such as callose, lignin, or hydroxyproline-rich glycoproteins, can inhibit pathogen penetration and/or spreading. We have previously demonstrated that calli from tomato cultivars resistant in vivo to Fusarium oxysporum f.sp. lycopersici show a high amount of polysaccharides in vitro. The aim of the present work was to assess the possible role of polysaccharide content and/or synthetic capacity in determining the competence of plant cells for active defence. For this purpose, tomato cell clones with increased and decreased polysaccharide (FL⁺, FL^-) and callose (A⁺, A^-) content have been selected by means of specific stains as visual markers and tested for the effect of these changes on the extent of response to Fusarium. The analysis of several parameters known to be indicative of active defence (cell browning after elicitor treatment, peroxidase and -glucanase induction and inhibition of fungal growth in dual culture) clearly shows that FL⁺ and A⁺ clones have acquired an increased competence for the activation of defence response. The results are thoroughly discussed in terms of an evaluation of the relative importance of constitutive and/or inducible polysaccharide synthetic capacity for plant response to pathogens, and their possible regulation by plant physiological backgrounds.     

Pullulanases of alkaline and broad pH range from a newly isolated alkalophilicBacillus sp. S-1 and aMicrococcus sp. Y-1

Summary Two highly alkalophilic bacteria, and potent producers of alkaline pullulanase, were isolated from Korean soils. The two isolates, identified asBacillus sp. S-1 andMicrococcus sp. Y-1, grow on starch under alkaline conditions and effectively secrete extracellular pullulanases. The two isolates were extremely alkalophilic since bacterial growth and enzyme production occurred at pH values ranging from pH 6.0 to 12.0 forMicrococcus sp. Y-1 and pH 6.0 to 10.0 forBacillus sp. S-1. Both strains secrete enzymes that possess amylolytic and pullulanolytic acitivities. Extracellular crude enzymes of both isolates gave maltotriose as the major product formed from soluble starch and pullulan hydrolysis. Compared to other alkalophilic microbes such asMicrococcus sp. (0.57 units ml^–1),Bacillus sp. KSM-1876 (0.56 units ml^–1) andBacillus No. 202-1 (1.89 units ml^–1) these isolates secreted extremely high concentrations (7.0 units ml^–1 forBacillus sp. S-1 and 7.6 units ml^–1 forMicrococcus sp. Y-1) of pullulanases in batch culture. The pullulanase activities from both strains were mostly found in the culture medium (85–90%). The extracellular enzymes of both bacteria were alkalophilic and moderately thermoactive; optimal activity was detected at pH 8.0–10.0 and between 50 and 60°C. Even at pH 12.0, 65% of original Y-1 pullulanase activity and 10% of S-1 pullulanase activity remained. The two newly isolated strains had broad pH ranges and moderate thermostability for their enzyme activities. These result strongly indicate that these new bacterial isolates have potential as producers of pullulanases for use in the starch industry.     

Antagonistic effects of soil bacteria onFusarium oxysporum Schlecht f.sp.dianthii (Prill and Del.) Snyd. and Hans.

Summary Thirty two bacteria antagonistic to a number of phytopathogenic fungi were isolated from soil samples. One bacterial strain, designated as M 51, appeared to be particularly active towardsF. oxysporum f. sp.dianthii, in vitro andin vivo and it was inhibitoryin vitro to three otherFusarium spp. used. Tests to find if there was protection against fusarium wilt were carried out by three different methods of inoculation of the cuttings: a) dipping of cuttings for ten minutes in bacterial suspension; b) spraying of suspension on perlite where the rooted cuttings were planted; c) spraying the greenhouse bench rooting boxes, where the non-rooted cuttings were planted, with bacterial suspension. Following this all the cuttings were transplanted into soil naturally highly infested withFusarium oxysporum f. sp.dianthii (3000 units/g). Good protection against fusarium wilt was obtained for cuttings inoculated by method (b). However protection decreased gradually about 60 days after they were transplanted; both control and inoculated cuttings showed a comparable mortality rate. Method of inoculation and the development of the protective effect are discussed.     

Colonization of cucumber plants by the biocontrol fungus Clonostachys rosea f. catenulata

Clonostachys rosea f. catenulata (Gliocladium catenulatum) strain J1446 (formulated as Prestop WP) suppressed Fusarium root and stem rot caused by Fusarium oxysporum f. sp. radicis-cucumerinum (Forc) on cucumber plants grown hydroponically in rockwool medium. Sixty days following application at seeding, the biocontrol agent had proliferated through the rockwool blocks and was present on cucumber roots and the crown region of the stem at populations >1 × 10⁵ CFU/g fresh weight. Scanning electron micrographs showed that C. rosea had rapidly colonized the root surface and was associated with root hairs and epidermal cell junctions. Following transformation of the fungus with Agrobacterium tumefaciens strain AGL-1 containing the hygromycin resistance (hph) and β-glucuronidase (uidA) genes, blue-stained mycelia could be seen growing on the surface and within epidermal and cortical cells of roots, stems and shoots 3 weeks after treatment. Quantification of GUS activity by fluorometric assays showed that fungal biomass was highest in the roots and crown area, while the extent of colonization of upper stems and true leaves was variable. Higher population levels resulted following application to rockwool blocks compared to seed treatment. Application of C. rosea preceding inoculation with Forc significantly reduced pathogen populations on roots compared to plants inoculated with Forc alone. Colonization of infection sites in the root zone reduced pathogen development and disease incidence. Densities of the biocontrol agent appeared to increase in the presence of the pathogen.     

Control of Fusarium wilt in bittergourd and bottlegourd by biofumigation using mustard var. Monteverde

Fusarium wilt disease is a growing concern in cucurbit crops in the Philippines. Most often than not, farmers highly depend on commercial fungicides for control but these chemicals are very expensive and not environment-friendly. Biofumigation and green manuring using Brassica plants is a potential alternative for sustainable management of this destructive disease. A study was conducted to evaluate the efficacy of mustard var. Montevede as a biofumigant and green manure to control Fusarium wilt disease of bittergourd (Momordica charantia L.) and bottlegourd (Lagenaria siceraria (Mol.) Standl.). In vitro assay of mustard slurry resulted in 100% suppression of the mycelial growth of F. oxysporum f. sp. momordicae and F. oxysporum f. sp. lagenariae isolates after exposure to 5, 10 and 15?g of mustard slurry compared with the control. Similarly, incorporation of the macerated mustard leaves in the infested soil reduced Fusarium wilt incidence by 100% in bittergourd and bottlegourd. The effect of mustard was comparable to Bavistin^® fungicide both in vitro and in vivo.     

Specific detection of an oil-degrading bacterium, Corynebacterium sp. IC10, in sand microcosms by PCR using species-specific primers based on 16S rRNA gene sequences

A species-specific PCR technique to detect an oil-degrading bacterium, Corynebacterium sp. IC10, released into sand microcosms is described. PCR primers, specific to strain IC10, were designed based on 16S rRNA gene sequences and tested against both closely and distantly related bacterial strains using four primer combinations involving two forward and two reverse primers. Two sets of them were specific to the strain IC10 and Corynebacterium variabilis and one set was selected for further analysis. The PCR amplification was able to detect 1 pg template DNA of strain IC10 and 1.2×10⁴ c.f.u. of IC10 ml wet sand^–1 in the presence of 3×10⁸ Escherichia coli cells. In non-sterile sand microcosms seeded with the strain IC10, the sensitivity of detection decreased to 9.6×10⁵ c.f.u. ml wet sand^–1. The detection sensitivity thus depends on the complexity of background heterogeneous DNA of environmental samples. The assay is suitable for detection of Corynebacterium sp. IC10 in laboratory microcosms, however, cross reaction with non-oil degrading coryneforms may prohibit its use in uncharacterized systems.     

Dissolution of ferric phosphate by alfalfa (Medicago sativa L.) root exudates

Alfalfa (Medicago sativa L.) was grown in hydroponic culture to investigate adaptation to Fe-deficiency. Root exudates released into the nutrient solution from Fe-deficient plants were trapped and condensed on an amberlite XAD-4 resin column. The diethyl ether fraction of these exudates dissolved ferric phosphate remarkably. The dissolving capability was about 62 times higher than that of root exudates obtained from Fe-sufficient plants in complete nutrient solution. The Fe-dissolving compound was separated and identified. It was a new natural compound with molecular formula C₁₄H₁₀O₅ and was identified as 2-(3,5-dihydroxyphenyl)-5,6-dihydroxybenzofuran by means of mass spectrometry and ¹H-nuclear magnetic resonance. This new compound worked as a phytoalexin and inhibited completely the fungal growth of Fusarium oxysporum f. sp. phaseoli.     

A substance inducing teliospore production in wheat leaf rust,Puccinia recondita f. sp.tritici

A substance inducing teliospore production inPuccinia racondita f. sp.tritici was found in water and methanol extracts of wheat leaves with telia of the wheat leaf rust just before harvest time. Methanol (MeOH) and water extracts from uninfected wheat leaves also showed telia-inducing activity. However, the MeOH and water extracts from wheat leaves covered with telia showed much stronger activity than those from uninfected wheat leaves. We obtained a fraction (0.2 mg) showing activity at 2 ng/ml by purification of the water extract.     

Construction of a Tn5 derivative encoding bioluminescence and its introduction in Pseudomonas, Agrobacterium and Rhizobium

Summary A simple method based upon the use of a Tn5 derivative, Tn5-Lux, has been devised for the introduction and stable expression of the character of bioluminescence in a variety of gram-negative bacteria. In Tn5-Lux, the luxAB genes of Vibrio harveyi encoding luciferase are inserted on a SalI-BglII fragment between the kanamycin resistance (Km^r) gene and the right insertion sequence. The transposon derivative was placed on a transposition suicide vehicle by in situ recombination with the Tn5 suicide vector pGS9, to yield pDB30. Mating between Escherichia coli WA803 (pDB30) and a strain from our laboratory, Pseudomonas sp. RB100C, gave a Km^r transfer frequency of 10^-6 per recipient, a value 10 times lower than that obtained with the original suicide vehicle pGS9. Tn5-Lux was also introduced by insertion mutagenesis in other strains of gram-negative soil bacteria. The bioluminescence marker was expressed in the presence of n-decanal, and was monitored as chemiluminescence in a liquid scintillation counter. The recorded light intensities were fairly comparable among the strains, and ranged between 0.2 to 1.8x10⁶ cpm for a cell density of 10³ colony forming units/ml. Nodules initiated by bioluminescent strains of Rhizobium leguminosarum on two different hosts were compared for intensity of the bioluminescence they produced.     

Antagonistic effects of soil bacteria onFusarium oxysporum Schlecht f. sp.dianthi (Prill and Del.) Snyd. and Hans

Summary Fusarium oxysporum f. sp.dianthi, pathogenic on carnation plants is very sensitive toBacillus subtilis M51 inhibition.Fusarium oxysporum disease (fusariosis) is prevented for a period of two months after treatment of plants withBacillus subtilis M51. The persistence ofB. subtilis M51, marked for selenomycin resistance (MZ51) and inoculated on the roots of carnation cuttings was studied. Soil used was two types: naturally infested withFusarium oxysporum and free from this pathogen. Bacterial cells presence on the roots was detected by direct plating and the presence of the pathogen in the roots was investigated by histological assays. Evidence gathered by these procedures suggest that plant protection is dependent on the physical presence ofB. subtilis M51 cells on the roots.     

Isolation of Endophytic Actinomycetes From Roots and Leaves of Banana (Musa Acuminata) Plants and Their Activities Against Fusarium oxysporumf. sp. cubense

Two hundred and forty-two actinomycete strains were isolated from the interior of leaves and roots of healthy and wilting banana plants. Most of them were streptomycetes, Streptomyces griseorubiginosus-like strains were the most frequently isolated strains. Community analysis demonstrated increased actinomycete diversity in wilting leaves compared to that in healthy leaves, similar actinomycete communities were found in wilting and healthy roots. Screening of the isolates for antagonistic activity against Fusarium oxysporumf. sp. cubenserevealed that the proportion of antagonistic streptomycetes in healthy roots was higher than that in wilting roots (P < 0.01), but no difference was found between antagonistic strains isolated from healthy and wilting leaves. The potential biological control of Panama disease of banana by endophytic streptomycetes, especially Streptomyces griseorubiginosus-like strains was discussed.