Oxidative burst elicited by Bacillus mycoides isolate Bac J, a biological control agent, occurs independently of hypersensitive cell death in sugar beet

Response of sugar beet cultivars C40 and USH11 to syringe infiltration of live and dead Bacillus mycoides isolate Bac J, a biological control agent, and virulent and avirulent isolates of Erwinia carotovora pv. betavasculorum was measured by monitoring systemic acquired resistance control of Cercospora beticola, specific activity of chitinase and beta-glucanase, the oxidative burst, and hypersensitive cell death at the infiltration site. Priming sugar beet with B. mycoides Bac J (1 x 10(8) cells/ml) and avirulent isolates of E. carotovora pv. betavasculorum (1 x 10(6) cells/ml) reduced C. beticola symptoms by nearly 70% on distal, untreated leaves. Systemic resistance responses elicited by live B. mycoides Bac J and avirulent E. carotovora pv. betavasculorum isolates, measured by assays for chitinase and beta-glucanase, were statistically equivalent, and biphasic hydrogen peroxide production was observed. Although similar in timing, the second hydrogen peroxide burst was twofold lower for B. mycoides Bac J than for avirulent E. carotovora pv. betavasculorum. Hypersensitive cell death was elicited by avirulent E. carotovora pv. betavasculorum but not B. mycoides Bac J. An oxidative burst was elicited by spray-applied B. mycoides Bac J under both light and green light conditions, indicating that the signal produced by B. mycoides Bac J was not reliant on the stomata for entry into sugar beet. A working model for signal delivery and systemic resistance induction by B. mycoides Bac J in sugar beet is proposed.     

甜菜增产菌P10菌株的鉴定

在新疆甜菜块根皮层内分离并筛选到对甜菜具有增产增糖作用的优化菌株Ｐ＿（１０）。通过形态培养征和生理生化特性研究,将该菌株鉴定为短小芽孢杆菌（Ｂａｃｉｌｕｓｐｕｍｉｌｕｓ）。     

Biological control of cucumber and sugar beet damping-off caused by Pythium ultimum with bacterial and fungal antagonists

AIMS: Five bacterial strains belonging to Bacillus subtilis, Pseudomonas fluorescens and Ps. corrugata and two fungal strains belonging to Trichoderma viride and Gliocladium virens were evaluated for their efficacy in controlling sugar beet and cucumber damping-off caused by Pythium ultimum. METHODS AND RESULTS: The in vitro antagonistic activity of bacteria against various Pythium spp. was evaluated with dual cultures in various media. Pseudomonas strains inhibited the pathogen better than Bacillus strains. To identify potentially useful antagonist combinations, dual compatibility of antagonists was also evaluated, based on growth in two liquid media containing substrate previously used by other antagonists. Four pairs of bacteria were selected. Sugar beet damping-off biocontrol was attempted with bacterial seed treatments (individually and in pairs). Cucumber damping-off biocontrol was attempted with bacterial seed treatments and bacterial and fungal compost treatments. In sugar beet, satisfactory biocontrol was only achieved with Pseudomonas antagonists. Antagonist combinations did not show any superior biocontrol ability to individual antagonists and compatibility of bacteria in vitro did not correlate with compatibility in vivo. Bacterial seed treatments and fungal compost treatments failed to control cucumber damping-off. Better biocontrol in cucumber was achieved when bacterial antagonists were applied by drenching or by coating seed with bacteria in a peat carrier. CONCLUSIONS: Pseudomonas antagonists were superior to Bacillus antagonists in controlling damping-off in cucumber and sugar beet. Pseudomonas peat inocula maintained a good shelf-life 2 years after preparation. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas peat formulations have the potential for development into commercial biopesticides.     

天山北坡甜菜内生菌分离鉴定及其动态变化

对新疆昌吉和石河子两地种植的甜菜内生菌进行了分离、鉴定和分析,结果表明甜菜内生菌多属于细菌,其中假单胞菌 (Pseudomonas sp. )和芽孢菌类(Bacillus sp.)的分离频率分别在33.2%～59.2%和12.7%～28.1%,是甜菜植株中的优势内生菌群.16S rDNA 和 ITS 序列同源性比较和系统发育分析表明内生菌具有丰富的多样性.根中内生菌的多样性高于茎、叶,昌吉地区种植的甜菜中分离出的内生菌种类较多.从感病品种及生长不良甜菜植株中分离出的内生菌种类比较丰富.通过回接分离及利用扫描电镜观察内生菌在植物体内分布发现,内生菌能够定殖于甜菜块根.     

Systemic resistance induced by Bacillus lipopeptides in Beta vulgaris reduces infection by the rhizomania disease vector Polymyxa betae

The control of rhizomania, one of the most important diseases of sugar beet caused by the Beet necrotic yellow vein virus, remains limited to varietal resistance. In this study, we investigated the putative action of Bacillus amylolequifaciens lipopeptides in achieving rhizomania biocontrol through the control of the virus vector Polymyxa betae. Some lipopeptides that are produced by bacteria, especially by plant growth-promoting rhizobacteria, have been found to induce systemic resistance in plants. We tested the impact of the elicitation of systemic resistance in sugar beet through lipopeptides on infection by P. betae. Lipopeptides were shown to effectively induce systemic resistance in both the roots and leaves of sugar beet, resulting in a significant reduction in P. betae infection. This article provides the first evidence that induced systemic resistance can reduce infection of sugar beet by P. betae.     

Suppression of sugar beet damping-off caused by Rhizoctonia solani using bacterial and fungal antagonists

Rhizoctonia damping-off caused by Rhizoctonia solani Kühn, is one of the most damaging sugar beet diseases. It causes serious economic damage wherever sugar beets are grown. Biological control is an efficient and environmentally friendly way to prevent damping-off disease. Suppression of damping-off disease caused by R. solani was carried out by four isolates of Bacillus subtilis (Ehrenberg) Cohn as well as three isolates of each of Trichoderma harzianum Rifai and Trichoderma hamatum (Bonord.) Bainier. The effect of Bacillus and Trichoderma isolates against R. solani was investigated in vitro and tested on sugar beet plants under greenhouse conditions. Isolates of Bacillus and Trichoderma were able to inhibit the growth of R. solani in dual culture. Furthermore, Trichoderma isolates gave high antagonistic effect than isolates of B. subtilis. Under greenhouse conditions, coating seeds by T. harzianum and B. subtilis separately, reduced seedling damping-off significantly. However, applications of T. harzianum increased the percentage of surviving plants more than B. subtilis in comparison to control. The obtained results indicate that T. harzianum and B. subtilis are very effective biocontrol agents that offer potential benefit in sugar beet damping-off and should be harnessed for further biocontrol applications.     

The autolytic phenotype of the Bacillus cereus group

AIM: To determine the autolytic phenotype of five species in the Bacillus cereus group. METHODS AND RESULTS: The autolytic rate of 96 strains belonging to five species in the B. cereus group was examined under starvation conditions at pH 6, 6.5 and 8.5 in different buffers. The autolytic rate was strain-dependent with a wide variability at pH 6, but higher and more uniform at pH 6.5. At pH 8.5, and respect to the extent of autolysis at pH 6.5, it was relatively low for most of the strains with the lowest values between 13 and 52% in Bacillus mycoides and Bacillus pseudomycoides. Peptidoglycan hydrolase patterns evaluated by renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis using cells of Bacillus thuringiensis ssp. tolworthi HD125 as an indicator, revealed complex profiles with lytic bands of about 90, 63, 46, 41, 38, 32, 28 and 25 kDa in B. cereus, B. thuringiensis and Bacillus weihenstephanensis. Bacillus mycoides and B. pseudomycoides had simpler profiles with lytic bands of 63, 46 and 38 kDa. Changes in the autolytic pattern were observed for cells harvested at the stationary phase of growth (72 h) showing an increase in the intensity of the 25 kDa band in the case of B. cereus, B. thuringiensis and B. weihenstephanensis, while no changes were observed for B. mycoides. Using Micrococcus lysodeicticus and Listeria monocytogenes as indicators lytic activity was retained by proteins of 63, 46, 38, 32 and 25 kDa and a new one of about 20 kDa in B. mycoides. Growth in the different media did not affect the autolytic pattern. NaCl abolished the activity of all the peptidoglycan hydrolases except for those of B. mycoides and B. weihenstephanensis. Lytic activity was retained in the presence of MgCl(2), MnCl(2) and EDTA and increased at basic pH. CONCLUSIONS: Bacillus cereus/B. thuringiensis/B. weihenstephanensis showed a high extent of autolysis around neutral pH, even though they presented relatively complex autolysin profiles at alkaline pH. Bacillus mycoides/B. pseudomycoides had a higher extent of autolysis at acidic pH and a simpler autolysin pattern. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on the autolytic phenotype expand the phenotypic characterization of the different species in the B. cereus group.     

Purification and characterization of thermostable endo-1,5-alpha-L-arabinase from a strain of Bacillus thermodenitrificans

A strain of a thermophilic bacterium, tentatively designated Bacillus thermodenitrificans TS-3, with arabinan-degrading activity was isolated. It produced an endo-arabinase (ABN) (EC 3.2.1.99) and two arabinofuranosidases (EC 3.2.1.55) extracellularly when grown at 60 degrees C on a medium containing sugar beet arabinan. The ABN (tentatively called an ABN-TS) was purified 7,417-fold by anion-exchange, hydrophobic, size exclusion, and hydroxyapatite chromatographies. The molecular mass of ABN-TS was 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the isoelectric point was pH 4.5. The enzyme was observed to be more thermostable than known ABNs; it had a half-life of 4 h at 75 degrees C. The enzyme had optimal activity at 70 degrees C and pH 6.0. The enzyme had apparent K(m) values of 8.5 and 45 mg/ml and apparent V(max) values of 1.6 and 1.1 mmol/min/mg of protein against debranched arabinan (alpha-1,5-arabinan) and arabinan, respectively. The enzyme had no pectin-releasing activity (protopectinase activity) from sugar beet protopectin, differing from an ABN (protopectinase-C) from mesophilic Bacillus subtilis IFO 3134. The pattern of degradation of debranched arabinan by ABN-TS indicated that the enzyme was an endo-acting enzyme and the main end products were arabinobiose and arabinose. The results of preliminary experiments indicated that the culture filtrate of strain TS-3 is suitable for L-arabinose production from sugar beet pulp at high temperature.     

Detection of antibiotic-related genes from bacterial biocontrol agents with polymerase chain reaction

Pseudomonas chlororaphis PA23, Pseudomonas spp. strain DF41, and Bacillus amyloliquefaciens BS6 consistently inhibit infection of canola petals by Sclerotinia sclerotiorum in both greenhouse and field experiments. Bacillus thuringiensis BS8, Bacillus cereus L, and Bacillus mycoides S have shown significant inhibition against S. sclerotiorum on plate assays. The presence of antibiotic biosynthetic or self-resistance genes in these strains was investigated with polymerase chain reaction and, in one case, Southern blotting. Thirty primers were used to amplify (i) antibiotic biosythetic genes encoding phenazine-1-carboxylic acid, 2,4-diacetylphloroglucinol, pyoluteorin, and pyrrolnitrin, and (ii) the zwittermicin A self-resistance gene. Our findings revealed that the fungal antagonist P. chlororaphis PA23 contains biosynthetic genes for phenazine-1-carboxylic acid and pyrrolnitrin. Moreover, production of these compounds was confirmed by high performance liquid chromatography. Pseudomonas spp. DF41 and B. amyloliquefaciens BS6 do not appear to harbour genes for any of the antibiotics tested. Bacillus thuringiensis BS8, B. cereus L, and B. mycoides S contain the zwittermicin A self-resistance gene. This is the first report of zmaR in B. mycoides.     

A randomly amplified polymorphic DNA marker specific for the Bacillus cereus group is diagnostic for Bacillus anthracis

Aiming to develop a DNA marker specific for Bacillus anthracis and able to discriminate this species from Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides, we applied the randomly amplified polymorphic DNA (RAPD) fingerprinting technique to a collection of 101 strains of the genus Bacillus, including 61 strains of the B. cereus group. An 838-bp RAPD marker (SG-850) specific for B. cereus, B. thuringiensis, B. anthracis, and B. mycoides was identified. This fragment included a putative (366-nucleotide) open reading frame highly homologous to the ypuA gene of Bacillus subtilis. The restriction analysis of the SG-850 fragment with AluI distinguished B. anthracis from the other species of the B. cereus group.     

Analysis of gene inheritance and expression in hybrids between transgenic sugar beet and wild beets

Reciprocal gene exchange between cultivated sugar beet and wild beets in seed production areas is probably the reason for the occurence of weed beets in sugar beet production fields. Therefore, when releasing transgenic sugar beet plants into the environment, gene transfer to wild beets ( Beta vulgaris ssp. maritima ) has to be considered. In this study the transfer of BNYVV- (beet necrotic yellow vein virus) resistance and herbicide-tolerance genes from two transgenic sugar beet lines that were released in field experiments in 1993 and 1994 in Germany to different wild beet accessions was investigated. In order to evaluate the consequences of outcrossing, manual pollinations of emasculated wild beet plants with homozygous transgenic sugar beet plants were performed. In the resulting hybrids the transgenes were stably inherited according to Mendelian law. Gene expression in leaves and roots of the hybrids was in the same range as in the original transgenic sugar beet plants. Moreover, it was found that in one of the wild beet accessions, transfer and expression of the BNYVV resistance gene did considerably increase the level of virus resistance.     

超表达AVP1基因提高甜菜的耐低磷和耐盐抗旱性

为探讨H⁺-焦磷酸酶编码基因对甜菜磷吸收和抗性的影响,实现优良基因在甜菜基因工程中的利用,研究在甜菜中超表达拟南芥液泡膜H⁺-焦磷酸酶编码基因AVP1,对转基因甜菜分析其耐低磷、耐盐性和抗旱性。结果显示,AVP1基因在甜菜植株的叶片和块根中表达,且在逆境胁迫下增强表达量响应胁迫;低磷处理条件下,转基因甜菜与野生型甜菜相比具有更高的含磷量,可提高甜菜对磷的吸收利用效率;干旱、盐胁迫处理条件下,AVP1基因在转基因甜菜中显著上升,在盐胁迫或干旱处理条件下,转基因植株的生长受抑程度相对较轻。随着盐和干旱胁迫的加剧,转基因植株体内MDA含量与野生型植株相比较低而脯氨酸含量显著增加,AVP1基因可通过减轻逆境对甜菜细胞膜的损伤及提高甜菜细胞的渗透调节能力,进而增强甜菜对高盐和干旱胁迫的抗性。     

Interactions of Bacillus spp. and plants--with special reference to induced systemic resistance (ISR)

Biological control of soil-borne pathogens comprises the decrease of inoculum or of the disease producing activity of a pathogen through one or more mechanisms. Interest in biological control of soil-borne plant pathogens has increased considerably in the last few decades, because it may provide control of diseases that cannot or only partly be managed by other control strategies. Recent advances in microbial and molecular techniques have significantly contributed to new insights in underlying mechanisms by which introduced bacteria function. Colonization of plant roots is an essential step for both soil-borne pathogenic and beneficial rhizobacteria. Colonization patterns showed that rhizobacteria act as biocontrol agents or as growth-promoting bacteria form microcolonies or biofilms at preferred sites of root exudation. Such microcolonies are sites for bacteria to communicate with each other (quorum sensing) and to act in a coordinated manner. Elicitation of induced systemic resistance (ISR) by plant-associated bacteria was initially demonstrated using Pseudomonas spp. and other Gram-negative bacteria. Several strains of the species Bacillus amyloliquefaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B. mycoides, and B. sphaericus elicit significant reductions in the incidence or severity of various diseases on a diversity of hosts. Elicitation of ISR by these strains has been demonstrated in greenhouse or field trials on tomato, bell pepper, muskmelon, watermelon, sugar beet, tobacco, Arabidopsis sp., cucumber, loblolly pine, and two tropical crops (long cayenne pepper and green kuang futsoi). Protection resulting from ISR elicited by Bacillus spp. has been reported against leaf-spotting fungal and bacterial pathogens, systemic viruses, a crown-rotting fungal pathogen, root-knot nematodes, and a stem-blight fungal pathogen as well as damping-off, blue mold, and late blight diseases. This progress will lead to a more efficient use of these strains which is worthwhile approach to explore in context of biocontrol strategies.     

Glycon specificity profiling of alpha-glucosidases using monodeoxy and mono-O-methyl derivatives of p-nitrophenyl alpha-D-glucopyranoside

Hydrolysis of probe substrates, eight possible monodeoxy and mono-O-methyl analogs of p-nitrophenyl alpha-D-glucopyranoside (pNP alpha-D-Glc), modified at the C-2, C-3, C-4, and C-6 positions, was studied as part of investigations into the glycon specificities of seven alpha-glucosidases (EC 3.2.1.20) isolated from Saccharomyces cerevisiae, Bacillus stearothermophilus, honeybee (two enzymes), sugar beet, flint corn, and Aspergillus niger. The glucosidases from sugar beet, flint corn, and A. niger were found to hydrolyze the 2-deoxy analogs with substantially higher activities than against pNP alpha-D-Glc. Moreover, the flint corn and A. niger enzymes showed hydrolyzing activities, although low, for the 3-deoxy analog. The other four alpha-glucosidases did not exhibit any activities for either the 2- or the 3-deoxy analogs. None of the seven enzymes exhibited any activities toward the 4-deoxy, 6-deoxy, or any of the methoxy analogs. The hydrolysis results, with the deoxy substrate analogs, demonstrated that alpha-glucosidases having remarkably different glycon specificities exist in nature. Further insight into the hydrolysis of deoxyglycosides was obtained by determining the kinetic parameters (k(cat) and K(m)) for the reactions of sugar beet, flint corn, and A. niger enzymes.     

Growth and photosynthetic efficiency promotion of sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.) by endophytic bacteria

Very little is known about the physiological interactions between plants and endophytic bacteria. We investigated the impact of three endophytic bacteria, Bacillus pumilus 2-1, Chryseobacterium indologene 2-2, and Acinetobacter johnsonii 3-1, on the photosynthetic capacity and growth of sugar beet. Endophyte-free plants were obtained first and infected with the bacteria. Measurements of total chlorophyll content revealed very significant differences between endophyte-free beet plants and some infected by endophytic bacteria. The maximum photochemical yield (Fv/Fm) was used to determine any photosynthetic effect on plants caused by biotic or abiotic factors. After 30 days of growth, there was significantly higher Fv/Fm for endophyte-infected than endophyte-free plants. The light response curves of beet showed that photosynthetic capacity was significantly increased in endophyte-infected plants. Photosynthesis of endophyte-free plants was saturated at 1,300 μmol m⁻² s⁻¹, whereas endophyte-infected plants were not saturated at the irradiance used. The effect seemed to be due to promotion of electron transport in the thylakoid membranes. Promotion of photosynthetic capacity in sugar beet was due to increased chlorophyll content, leading to a consequent increased carbohydrate synthesis. It is possible that the increased maximum yield of photosynthesis in sugar beet was promoted by phytohormones and produced by the bacteria.     

A comparative study of enzyme variation in Bacillus cereus and Bacillus thuringiensis

Thirty-two strains of Bacillus spp. were examined in a multilocus enzyme study by agarose gel electrophoresis. The organisms were Bacillus thuringiensis (21 strains), B. cereus (8), including two of var. mycoides , and B. megaterium (3). Strains having similar enzyme variants were grouped into zymovars. A total of 10 of 11 enzyme loci studied were polymorphic and 27 zymovars were distinguished among the 32 strains. The results were subjected to numerical analysis, phenetic affinities and genetic distances between the strains were calculated. The numerical analysis was unable to differentiate between B. thuringiensis and B. cereus . Our results indicated that based on this multilocus enzyme study these zymovars should be considered as belonging to the same species. A mycoides variant of B. cereus was the most distinctive strain studied and clearly belonged to a separate species, B. mycoides. The technique also allowed for identification of contamination and mislabelling of strains.     

A comparative study of enzyme variation in Bacillus cereus and Bacillus thuringiensis

Thirty-two strains of Bacillus spp. were examined in a multilocus enzyme study by agarose gel electrophoresis. The organisms were Bacillus thuringiensis (21 strains, B. cereus (8), including two of var. mycoides, and B. megaterium (3). Strains having similar enzyme variants were grouped into zymovars. A total of 10 of 11 enzyme loci studied were polymorphic and 27 zymovars were distinguished among the 32 strains. The results were subjected to numerical analysis, phenetic affinities and genetic distances between the strains were calculated. The numerical analysis was unable to differentiate between B. thuringiensis and B. cereus. Our results indicated that based on this multilocus enzyme study these zymovars should be considered as belonging to the same species. A mycoides variant of B. cereus was the most distinctive strain studied and clearly belonged to a separate species, B. mycoides. The technique also allowed for identification of contamination and mislabelling of strains.     

Mitochondrial minisatellite polymorphisms in fodder and sugar beets reveal genetic bottlenecks associated with domestication

Historically, sugar beets were selected from fodder beets. We used mitochondrial minisatellite loci to analyze cytoplasmic genetic diversity in fodder beet and sugar beet. Among the 8 sugar beet accessions examined we identified 3 multi-locus haplotypes. These 3 haplotypes were a subset of 5 haplotypes identified among the 29 fodder beet accessions examined. All but one haplotype in fodder beet comprised, in turn, a subset of 12 haplotypes identified previously in leaf beets. Such apparent decreases in cytoplasmic genetic diversity must result from genetic bottlenecks associated with domestication and the ensuing breeding processes. We also detected the haplotype associated with the male-sterile Owen cytoplasm of sugar beet in the fodder beet gene pool. Furthermore, the presence of a 39 kDa protein associated with the Owen cytoplasm was confirmed in two fodder beet plants by Western blot analysis. These results lead us to speculate that the Owen cytoplasm may have originated in fodder beet, from which sugar beet was derived.     

Bodenbewohnende Schädlinge und Seitenwurzelfäule der Zuckerrübe in Abhängigkeit von der Rotation – Ergebnisse eines Fruchtfolgeversuches

Soil inhabiting pests and rot of feeding roots of sugar beet depending on rotation - Results of a long-term trial Over a period of 17 years a trial was carried out with sugar beet, cereals and oilseed rape in different crop rotations on a field near Göttingen (Lower Saxony). The frequency of sugar beet in the rotation was 17, 25, 33 and 67 %. In absence of beet nematodes, root and sugar yield of the beet decreased after repeated growing of sugar beet in short rotations compared to variants with long rotations. Sugar content and beet quality were only slightly influenced. By applying a bioassay (BW-Test) with young beet plants in the greenhouse it was shown that increasing infections on the tips of rootlets of the beet plants were the cause for decreasing beet yield in close rotations. Mortality of young beet plants and progress of infection in the test indicated roughly the quantity of pathogenic fungi in the soil. In the roots of the bait platits the parasitic fungus Aphanomyces cochlioides predominated. Rate of infection and yield reduction in the field were decisively influenced by weather conditions. Differences in yield between sugar beet grown in a three-year and a four-year rotation, however, were not significant. An occurrence of beet pests depending on crop rotation was stated only for Atomaria linearis and this only in a few years.