枯草芽孢杆菌对几种灰霉病菌的抑制效果研究

分别研究了枯草芽孢杆菌(BacillussubtilisCohn)培养液、过滤液和灭活液对葡萄灰霉病菌(GB)、草莓灰霉病菌(SB)、辣椒灰霉病菌(PB)和番茄灰霉病菌(TB)菌丝生长的抑制作用。结果表明:枯草芽孢杆菌培养液对GB、SB、PB和TB都有很好的抑制作用。在菌液浓度达到10 5CFU/mL时,对4种灰霉病菌的抑制率均达到了10 0 % ;当浓度降低为10 4CFU/mL时,抑制率明显降抵。而菌液浓度为10 8CFU/mL时的过滤液,对GB、PB和TB的抑制率也均在5 0 %以上。灭活液对灰霉菌的抑制作用明显减弱,菌液浓度为10 8CFU/mL时,对PB、GB、TB和SB的抑制率分别为73.6 %、39.5 %、5 0 %和2 5 %。     

Trichoderma harzianum T39 Preparation for Biocontrol of Plant Diseases-Control of Botrytis cinerea , Sclerotinia sclerotiorum and Cladosporium fulvum

Isolate T39 of Trichoderma harzianum (TRICHODEX) is a commercial biocontrol agent. It controls Botrytis cinerea (grey mould) in greenhouse crops and in vineyards, Sclerotinia sclerotiorum (white mould) in various greenhouse and field crops, Cladosporium fulvum (leaf mould) in tomato, and the powdery mildews Sphaerotheca fusca in cucurbits and Leveillula taurica in pepper. T. harzianum T39 was applied in vineyards and greenhouses as part of grey mould management programmes in alternation with chemical fungicides. In the present study, the effect of T39 on diseases of greenhouse crops was demonstrated. The biocontrol agent was applied in formulations containing two concentrations of the active ingredient, or in the presence of oil in cucumber and tomato greenhouses. Suppression of B. cinerea , C. fulvum and S. sclerotiorum was similar when T39 was applied at final active ingredient rates of 0.2 or 0.4 g l -1 , except for one sampling date in one experiment. The addition of JMS Stylet-Oil did not contribute to the control of the above mentioned diseases achieved by T39.     

Ability of Clonostachys rosea to Establish and Suppress Sporulation Potential of Botrytis cinerea in Deleafed Stems of Hydroponic Greenhouse Tomatoes

The ability of Clonostachys rosea to establish and persist in deleafed tomato stems and to suppress sporulation potential of Botrytis cinerea was investigated in plots of hydroponic tomatoes in commercial greenhouses. Leaves near lower fruit clusters were removed according to standard practice and deleafed portions of the stems were treated with C. rosea , iprodione or water. Inoculum of B. cinerea was from natural infections. Stem lesions were not produced by the pathogen during the trials. Development of C. rosea and B. cinerea in stems was estimated indirectly by quantifying sporulation on excised stem tissues that were incubated on an agar medium containing paraquat. Incidence and area of sporulation of C. rosea on tissue pieces were high (76-99%) and moderately high (33-79%), respectively, when stems were treated with the agent at 0, 6, 24 or 48 h after deleafing and sampled 11 to 75 days later. In various instances, the agent also sporulated on tissues from water controls and iprodione treatments, apparently after interplot transmission. In most instances, incidence and area of sporulation of B. cinerea on tissue pieces were high (83-100%) and moderate to high (35-76%), respectively, in the water controls, but moderate (31-44%) and moderate to low (5-34%), respectively, for stems treated with C. rosea at 0 to 48 h after deleafing and sampled after 11-75 days. Without exception, C. rosea suppressed B. cinerea as or more effectively than iprodione. Correlations between inoculum density of C. rosea (0-10 6 conidia mL -1 ) and sporulation potential of B. cinerea in deleafed stems were strongly negative in each of three tests ( r = -0.95 to -0.99). Conidial suspensions and a talc formulation of C. rosea were of similar effectiveness against B. cinerea . We conclude that C. rosea persisted and suppressed sporulation potential of B. cinerea in deleafed tomato stems for at least 11 weeks after application.     

Potential use and mode of action of the new strain Bacillus thuringiensis UM96 for the biological control of the grey mould phytopathogen Botrytis cinerea

The potential use of Bacillus thuringiensis UM96 as a biocontrol agent for the grey mould phytopathogen Botrytis cinerea was evaluated. In order to dissect the mode of action of this UM96 strain, we also examined the role of lytic activities in the antagonism. First, B. thuringiensis UM96 was characterised based on 16S rRNA and gyrA gene sequencing and phenotypic traits. Petri dish biocontrol assays demonstrated that when strain UM96 was inoculated 24 h previous to B. cinerea, the mycelial growth was inhibited by up to 70%. Test for lytic enzymes activities of cellulase and glucanase was negative. Chitinase was the only positive enzyme activity in two different culture media. PCR detection of the chiB gene was also positive. Chitinolytic supernatants, obtained from rich and minimal media supplemented with colloidal chitin as the sole carbon source, from B. thuringiensis UM96 showed a strong inhibitory effect of B. cinerea that was not observed with heat-treated supernatant. Interestingly, when the supernatant was supplemented with 100 µM allosamidin, a chitinase specific inhibitor, the antagonistic activity was suppressed significantly. A lack of chitinase activity was also observed in allosamidin-treated supernatants. Our pathogenic B. cinerea strain also exhibited susceptibility to pure Streptomyces griseus chitinase. Finally, the chitinolytic strain B. thuringiensis UM96 was able to protect Medicago truncatula plants in vitro from B. cinerea infection and significantly reduced the necrotic zones and root browning of the plants. Together, these results suggest a potential use of B. thuringiensis UM96 for the biological control of B. cinerea and a role for chitinases during the antagonism displayed.     

Effect of cultivation conditions on spore production from Bacillus amyloliquefaciens B128 and its antagonism to Botrytis elliptica

Aims:  To maximize spore production by Bacillus amyloliquefaciens B128, and its antagonism to the fungal pathogen Botrytis elliptica B061. Methods and Results:  In the 5-l stirred-tank bioreactor (STR), with the 0·5 vvm aeration rate, an agitation rate of 200 rev min⁻¹ significantly enhanced the spore yield compared to the same in 300 rev min⁻¹ cultivations. In a 20-l airlift bioreactor (ALR) the maximal spore production was further increased with a controlled aeration rate of 2·5 vvm operated in a 24-mesh net-draft tube mode, and no pH control cultivation. This spore yield in the 20-l ALR was five- and eightfold higher; in addition the cultivation period was 19 h shorter, compared to that obtained from shaker flask and in the 5-l STR cultivations respectively. Conclusions:  Although culture conditions are still to be optimized, by using an ALR with net-draft tube, a scaling up from shaker flasks and STR to ALR of spore production by the strain B128 is technically feasible. Significance and Impact of the Study:  The spore yields obtained using bioreactors were much higher than those previously reported. The freshly produced spore preparations from the B128 strain significantly antagonized the grey mould pathogen B. elliptica.     

Role of lipopeptides produced by Bacillus subtilis GA1 in the reduction of grey mould disease caused by Botrytis cinerea on apple

AIM: Test of Bacillus subtilis strain GA1 for its potential to control grey mould disease of apple caused by Botrytis cinerea. METHODS AND RESULTS: GA1 was first tested for its ability to antagonize in vitro the growth of a wide variety of plant pathogenic fungi responsible for diseases of economical importance. The potential of strain GA1 to reduce post-harvest infection caused by B. cinerea was tested on apples by treating artificially wounded fruits with endospore suspensions. Strain GA1 was very effective at reducing disease incidence during the first 5 days following pathogen inoculation and a 80% protection level was maintained over the next 10 days. Treatment of fruits with an extract of GA1 culture supernatant also exerted a strong preventive effect on the development of grey mould. Further analysis of this extract revealed that strain GA1 produces a wide variety of antifungal lipopeptide isomers from the iturin, fengycin and surfactin families. A strong evidence for the involvement of such compounds in disease reduction arose from the recovery of fengycins from protected fruit sites colonized by bacterial cells. CONCLUSIONS: The results presented here demonstrate that, despite unfavourable pH, B. subtilis endospores inoculated on apple pulp can readily germinate allowing significant cell populations to establish and efficient in vivo synthesis of lipopeptides which could be related to grey mould reduction. SIGNIFICANCE AND IMPACT OF THE STUDY: This work enables for the first time to correlate the strong protective effect of a particular B. subtilis strain against grey mould with in situ production of fengycins in infected sites of apple fruits.     

Effects of ethylene biosynthesis in carrot root slices on 6-methoxymellein accumulation and resistance to Botrytis cinerea

The role of ethylene biosynthesis in the resistance response of carrot ( Daucus carota L., cv. Chantenay red-cored) slices to infection by Botrytis cinerea Pers. ex Pers. was investigated using aminoethoxyvinylglycine (AVG) and inhibitor of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (EC 4.4.1.-), and norbornadiene, an inhibitor of ethylene binding. Carrot slices became susceptible to a normally non-invasive level (10⁵ ml^-1) of spores of B. cinerea after treatment with AVG. ACC partially reversed the susceptibility induced by AVG. The ability of a crude pectic enzyme preparation from B. cinerea to induce resistance to a normally invasive level of B. cinerea spores (10⁶ ml^-1) was prevented by AVG. Accumulation of the carrot phyto-alexin 6-methoxymellein (6-MM) was prevented by norbornadiene, but it had no effect on the resistance response. An event associated with ethylene biosynthesis other than 6-MM accumulation appears to be responsible for the resistance of carrot slices to infection by B. cinerea.     

Inhibitory effects on microbial growth of Botrytis cinerea and Erwinia carotovora on potato using of a biopolymer chitosan at different molecular weights

The antimicrobial efficiency of chitosan at different molecular weights (5, 37, 57 and 290 kDa) against Botrytis cinerea and Erwinia carotovora on potato (Solanum tuberosum L.) was investigated. In vitro study showed that chitosan of 37 kDa was the most active against E. carotovora (minimum inhibitory concentration (MIC) = 950 mg/L), whereas 5 kDa chitosan was the most active against B.cinerea. Coating of potato tubers with 100, 250 and 500 mg/L significantly decreased the rate of weight loss and chitosan of 37 kDa showed the best effect. The in vivo antibacterial effect indicated that all treatments (500, 1000 and 2000 mg/L) significantly inhibited the growth of E. carotovora compared with the control. The lowest decay incidence was observed with 37 kDa chitosan. However, the antifungal activity against B. cinerea inoculated of leaves showed no decay incidence at 500 and 1000 mg/L with 57 kDa chitosan after 48 h.     

一株产木聚糖酶的蜡样芽孢杆菌对雪茄烟叶成分及发酵产物的影响

雪茄烟叶中的半纤维素在叶脉及叶片韧性等特性中发挥重要作用,烟叶叶脉较粗,半纤维素含量过高,导致雪茄烟叶韧性较差,可用性变差.本实验室前期筛选到一株产木聚糖酶的蜡样芽孢杆菌,该菌株对烟碱有较好的耐受性,利用液态发酵方法研究菌株以雪茄烟叶为营养源产木聚糖酶的最佳发酵条件,并对最佳发酵条件下菌株降解烟叶中半纤维素的效果进行测...     

Antagonistic effects of volatiles generated by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> on spore germination and hyphal growth of the plant pathogen, <Emphasis Type="Italic">Botrytis cinerea</Emphasis>

Botrytis cinerea is one of the most serious post-harvest pathogens of fruits and vegetables. Volatiles generated by Bacillus subtilis JA significantly inhibited both spore germination and elongation of germ tubes in Botrytis cinerea using a two-compartment agar-plate assay. The volatiles caused protoplasm retraction from the hyphal tips to the spores. Hua Chen and Xiang Xiao have contributed equally to this work.     

Optimization of cell growth and poly(3-hydroxybutyrate) accumulation on date syrup by a Bacillus megaterium strain

Optimal growth and PHB accumulation in Bacillus megaterium occurred with 5% (w/v) date syrup or beet molasses supplemented with NH₄Cl. When date syrup and beet molasses were used alone without an additional nitrogen source, a cell density of about 3gl^–1 with a PHB content of the cells of 50% (w/w) was achieved. NH₄NO₃ followed by ammonium acetate and then NH₄Cl supported cell growth up to 4.8gl^–1, whereas PHB accumulation was increased with NH₄Cl followed by ammonium acetate, NH₄NO₃ and then (NH₄)₂SO₄ to a PHB content of nearly 42% (w/w). Cultivation of B.megaterium at 30l scale gave a PHB content of 25% (w/w) of the cells and a cell density of 3.4gl^–1 after 14h growth.     

The effect of complex carbon and nitrogen, salt, Tween-80 and acetate on delta-endotoxin production by a Bacillus thuringiensis subsp kurstaki

Delta-endotoxin production by a strain of Bacillus thuringiensis subsp kurstakion complex media based on crude gruel and fish meal was investigated. High proteolytic activities were concomitantly produced with the bioinsecticide. In such complex media, the repressive regulation due to readily consumed carbon sources was partially overcome. In order to improve substrate assimilation, 0.5 g L⁻¹ sodium chloride and 0.1% Tween-80 were supplemented to the production medium, increasing delta-endotoxin yields when using gruel concentrations below 59 g L⁻¹. At and beyond 75 g L⁻¹ gruel, delta-endotoxin yields were not affected in the presence of 0.5 g L⁻¹ NaCl and 0.1% Tween-80, but proteolytic activity yields were remarkably reduced. Thus, the use of sodium chloride and Tween-80 allowed reduction of the initial gruel concentration to 42 g L⁻¹ for the production of 3350 mg L⁻¹ delta-endotoxin, while it was only 3800 mg L⁻¹ with 92 g L⁻¹ gruel. Moreover, similar to 0.5 g L⁻¹ NaCl and 0.1% Tween-80, the use of 10 g L⁻¹ sodium acetate significantly improved delta-endotoxin production and also reduced the proteolytic activity to 250 U ml⁻¹. Received 05 November 1998/ Accepted in revised form 19 August 1999     

Biotransformation of glycerol to 3-hydroxypropionaldehyde: Improved production by in situ complexation with bisulfite in a fed-batch mode and separation on anion exchanger

3-Hydroxypropionaldehyde (3HPA) is an important C3 chemical that can be produced from renewable glycerol by resting whole cells of Lactobacillus reuteri. However the process efficiency is limited due to substrate inhibition, product-mediated loss of enzyme activity and cell viability, and also formation of by-products. Complex formation of 3HPA with sodium bisulfite and subsequent binding to Amberlite IRA-400 was investigated as a means of in situ product recovery and for overcoming inhibition. The adsorption capacity and -isotherm of the resin were evaluated using the Langmuir model. The resin exhibited maximum capacity of 2.92 mmol complex/g when equilibrated with 45 mL solution containing an equilibrium mixture of 2.74 mmol 3HPA-bisulfite complex and 2.01 mmol free 3HPA. The dynamic binding capacity based on the breakthrough curve of 3HPA and its complex on passing a solution with 2.49 mmol complex and 1.65 mmol free 3HPA was 2.01 mmol/g resin. The bound 3HPA was desorbed from the resin using 0.20 M NaCl with a high purity as a mixture of complexed- and free 3HPA at a ratio of 0.77 mol/mol. Fed-batch biotransformation of glycerol (818.85 mmol) with in situ 3HPA complexation and separation on the bisulfite-functionalized resin resulted in an improved process with consumption of 481.36 mmol glycerol yielding 325.54 mmol 3HPA at a rate of 17.13 mmol/h and a yield of 68 mol%. Also, the cell activity was maintained for at least 28 h.