Three native Pseudomonas fluorescens strains tested under growth chamber and field conditions as biocontrol agents against damping-off in alfalfa

Alfalfa (Medicago sativa) is one of the most important crops used in Uruguay for livestock feeding. Seedling diseases, particularly damping-off, are a critical factor which limits its establishment. Three native Pseudomonas fluorescens strains, UP61.2, UP143.8 and UP148.2, previously isolated from Lotus corniculatus, were evaluated to determine their efficacy as biological control agents for alfalfa seedling diseases in the field. Their compatibility with the alfalfa-Sinorhizobium meliloti symbiosis was also assessed. In growth chamber conditions seed inoculation with Pseudomonas strains did not affect different parameters of alfalfa-rhizobium symbiosis as shown by nodulation rate and shoot dry weight of plants. The presence of the commercial inoculant strains of S. meliloti did not impair colonization by the P. fluorescens and vice versa. In field trials the dynamics of rhizobial rhizospheric populations were not affected by the presence of P. fluorescens. Each P. fluorescens strain successfully colonized alfalfa roots at adequate densities for biocontrol activity. Results showed that P. fluorescens strains provided a 10–13% increase in the number of established plants relative to the control, an intermediate result compared to the fungicide treatment (24%). The alfalfa above-ground biomass was increased by 13% and 15–18% in the presence of the fungicide and P. fluorescens strains, respectively. Therefore, results from this study demonstrated that the three P. fluorescens strains provided effective control against soil-borne pathogens and suggest a potential use in the development of a commercial inoculant to be applied for the control of legume seedling diseases.     

Osmotic stress adaptation, compatible solutes accumulation and biocontrol efficacy of two potential biocontrol agents on Fusarium head blight in wheat

Fusarium head blight (FHB) caused by Gibberella zeae (anamorph = Fusarium graminearum) is a devastating disease that causes extensive yield and quality losses to wheat in humid and semi-humid regions of the world. Biological control has been demonstrated to be effective under laboratory conditions but a few biocontrol products have been effective under field conditions. The improvement in the physiological quality of biocontrol agents may improve survival under field conditions, and therefore, enhance biocontrol activity. Bacillus subtilis RC 218 and Brevibacillus sp. RC 263 were isolated from wheat anthers and showed significant effect on control of FHB under greenhouse assays. This study showed the effect of water availability measured as water activity (a_W) using a growth medium modified with NaCl, glycerol and glucose on: (i) osmotic stress tolerance, (ii) viability in modified liquid medium, (iii) quantitative intracellular accumulation of betaine and ectoine and (iv) the biocontrol efficacy of the physiologically improved agents. Viability of B. subtilis RC 218 in NaCl modified media was similar to the control. Brevibacillus sp. RC 263 showed a limited adaptation to growth in osmotic stress. Betaine was detected in high levels in modified cells but ectoine accumulation was similar to the control cells. Biocontrol activity was studied in greenhouse assays on wheat inoculated at anthesis period with F. graminearum RC 276. Treatments with modified bacteria reduced disease severity from 60% for the control to below 20%. The physiological improvement of biocontrol agents could be an effective strategy to enhance stress tolerance and biocontrol activity under fluctuating environmental conditions.     

Biocontrol of tomato wilt by Penicillium oxalicum formulations in different crop conditions

Eight formulations of Penicillium oxalicum (FOR1 to FOR8) were obtained by the addition of various ingredients, in two separate steps of the production and drying of P. oxalicum conidia. These formulations were then evaluated against tomato wilt in three glasshouse (G1 to G3) and two field (F1 and F2) experiments. All formulations were applied to seedlings in seedbeds 7 days before transplanting at a rate of 10⁷ spores g⁻¹ seedbed substrate. The conidial viability of each formulation was estimated by measuring germination just after fluid bed-drying, before seedbed application and after 1 and 2 years of storage at 4 °C under vacuum. The densities of P. oxalicum were estimated in the seedbed substrate and in the rhizosphere of three plants per treatment just before transplanting. Initial conidial viability of formulations just after fluid bed-drying was approx. 80%, except for FOR1, FOR4, and FOR7 which were 60%. The initial viability was maintained up to 40–50% for 2 years of storage at 4 °C under vacuum, except for FOR1. All formulations had 50% viability at application time. Populations of P. oxalicum in the seedbed substrate just before transplanting were >10⁶ cfu g⁻¹ soil in G3 and F2; populations in rhizosphere were also >10⁶ cfu g⁻¹ fresh root, except for FOR3, FOR5, and FOR6 in G2. A range of 22–64% of disease reduction was observed with all formulations, although these reductions were not significant (p = 0.05) for FOR1, FOR4, and FOR5 in any experiment. Contrast analysis showed significant differences between biological treatments and untreated control (p = 0.05) in all experiments, but no significant differences between biological and chemical treatments. Initial conidial viability of P. oxalicum in formulations and populations of P. oxalicum in the seedbed substrate explained 78.26% of the variability in P. oxalicum populations in tomato rhizosphere before transplanting. Disease incidence in untreated plants was negatively correlated (r = −0.54) with the percentage of disease control. The relationship between the viability of formulations, the populations of P. oxalicum in seedbed and rhizosphere, and the control of tomato wilt is discussed.     

Rhizospheric bacteria as potential biocontrol agents against Fusarium wilt and crown and root rot diseases in tomato

The discovery of novel biocontrol agents requires the continuous scrutiny of native microorganisms to ensure that they will be useful on a regional scale. The goal of the present work was to discover novel antagonistic bacteria against Fusarium oxysporum ff. spp. lycopersici race 3 (Fol R3) and radicis-lycopersici (Forl) causing Fusarium wilt disease and Fusarium crown and root rot of tomatoes, respectively. High-throughput liquid antagonism screening of 1,875 rhizospheric bacterial strains followed by dual confrontation assays in 96-well plates was used to select bacteria exhibiting > 50% fungal growth inhibition. In a second dual confrontation assay in 10-cm Petri dishes, bacteria showing > 20% Fol R3 or Forl growth inhibition were further screened using a blood hemolysis test. After discarding β-hemolytic bacteria, a seedling antagonistic assay was performed to select five potential antagonists. A phylogenetic analysis of 16S rRNA identified one strain as Acinetobacter calcoaceticus (AcDB3) and four strains as members of the genus Bacillus (B. amyloliquefaciens BaMA26, Bacillus siamensis BsiDA2, B. subtilis BsTA16 and B. thuringiensis BtMB9). Greenhouse assays demonstrated that BsTA16 and AcDB3 were the most promising antagonists against Fol R3 and Forl, respectively. Pathogen biocontrol and growth promotion mechanisms used by these bacteria include the production of siderophores, biofilm, proteases, endoglucanases and indole acetic acid, and phosphate solubilization. These five bacteria exerted differential responses on pathogen control depending on the tomato hybrid, and on the growth stage of tomatoes. We report for the first time the use of an Acinetobacter calcoaceticus isolate (AcDB3) to control Forl in tomato under greenhouse conditions.     

Kinetics of production and characterization of the fucose-containing exopolysaccharide from Enterobacter A47

A fucose-containing exopolysaccharide (EPS) was produced by the bacterium Enterobacter A47 using glycerol byproduct from the biodiesel industry. The analysis of kinetic data suggested a partially growth associated EPS synthesis model. Although the EPS was composed of fucose, galactose and glucose at all cultivation stages, their relative proportion has varied considerably during the run. At the beginning (24 h), glucose was the main component (82.4 wt.%), being fucose and galactose minor components (5.0 wt.% and 10.9 wt.%, respectively), while at the end (96 h) it was composed of 26.0 wt.% fucose, 28.9 wt.% galactose and 43.7 wt.% glucose. The acyl groups content and composition have also changed, reaching their maximum content (19.2 wt.%) at the end of the run. Moreover, the molecular weight has increased linearly during the run (from 8 × 10⁵ to 5 × 10⁶). The changes observed in EPS composition and molecular weight have also had an impact upon the polymer's intrinsic viscosity, as shown by its linear increase from 3.95 to 10.72 dL g⁻¹. The results suggest that the culture might have synthesized at least two distinct EPS, with different sugar composition and average molecular weight, which predominated at different cultivation stages.     

Evaluation of rhizosphere bacterial antagonists for their potential to bioprotect potato (Solanum tuberosum) against bacterial wilt (Ralstonia solanacearum)

Bacterial wilt (Ralstonia solanacearum) is one of the production constraints of potato (Solanum tuberosum). The intent of the study was to evaluate potential of bacterial antagonists to suppress bacterial wilt disease development and evaluate the role of the strains as plant growth-promoting rhizobacteria (PGPR) in potato. One hundred-twenty rhizosphere bacterial isolates were screened against virulent strain of Ralstonia solanacearum PPRC-Rs. After in vitro screening, six antagonistic strains (PFMRI, BS-DFS, PF9, PF20, BC, and BS-wly) with inhibition diameter >11 mm were selected and studied further in the greenhouse, in vivo. During in vivo study, the strains were evaluated for their effect in suppressing disease development in terms of area under disease progress curve (AUDPC) and increasing biomass (plant height and dry weight) of potato. Accordingly, PFMRI, BS-DFS, and PF9, significantly reduced AUDPC by 78.6, 66, and 64.3%, and wilt incidence by 82.7, 66.2, and 65.7%, respectively, compared to the control. During the sole application, the strains significantly (P < 0.0001) increased plant height by 35.6, 45.9, and 45%, and dry matter by 111, 130.4, and 129%, respectively compared to non-bacterized control. In the presence of the pathogen strain PFMRI, BS-DFS, and PF9 increased plant height by 66, 50, and 48.2%, and dry matter by 153.8, 96.8, and 92.5%, respectively compared to the pathogen treated control. Hence, the study shows that PFMRI, BS-DFS, and PF9 strains have potential use in potato bioprotection, as PGPR or in an integrated bacterial wilt management; whose effectiveness under a variety of field conditions should be investigated.     

Evaluation of endophytic actinobacteria as antagonists of Gaeumannomyces graminis var. tritici in wheat

Endophytic actinobacteria isolated from healthy cereal plants were assessed for their ability to control fungal root pathogens of cereal crops both in vitro and in planta. Thirty eight strains belonging to the genera Streptomyces, Microbispora, Micromonospora, and Nocardioidies were assayed for their ability to produce antifungal compounds in vitro against Gaeumannomyces graminis var. tritici (Ggt), the causal agent of take-all disease in wheat, Rhizoctonia solani and Pythium spp. Spores of these strains were applied as coatings to wheat seed, with five replicates (25 plants), and assayed for the control of take-all disease in planta in steamed soil. The biocontrol activity of the 17 most active actinobacterial strains was tested further in a field soil naturally infested with take-all and Rhizoctonia. Sixty-four percent of this group of microorganisms exhibited antifungal activity in vitro, which is not unexpected as actinobacteria are recognized as prolific producers of bioactive secondary metabolites. Seventeen of the actinobacteria displayed statistically significant activity in planta against Ggt in the steamed soil bioassay. The active endophytes included a number of Streptomyces, as well as Microbispora and Nocardioides spp. and were also able to control the development of disease symptoms in treated plants exposed to Ggt and Rhizoctonia in the field soil. The results of this study indicate that endophytic actinobacteria may provide an advantage as biological control agents for use in the field, where others have failed, due to their ability to colonize the internal tissues of the host plant.     

番茄青枯雷尔氏菌强致病力菌株变异的研究

为研究番茄青枯雷尔氏菌强致病力菌株的变异,探索了继代培养、在NB培养基上不同时间培养、不同pH处理7d和15d、不同温度处理1h后对强致病力菌株变异的影响。结果表明:随着继代培养的培养代数增加,平均弱化指数成增大趋势,在第10代出现了无致病力菌株;在NB培养基上培养15d时,强致病力菌株已完全转化为不确定菌株和无致病力菌株,在培养30d时,强致病力菌株几乎完全转化为无致病菌株;pH7.0时,处理7d和15d后,强致病力菌株比例均为最大,分别为93.33%和92.22%,pH5.8时,强致病力菌株比例最低,分别为46.67%和31.11%;用不同温度处理强致病力菌株发现,温度50℃时,菌株死亡,温度40℃时,活菌数显著低于其他(4~30℃)处理,强致病力菌株比例为4~40℃所有处理中最低。     

Combined application of botanical formulations and biocontrol agents for the management of Fusarium oxysporum f. sp. cubense (Foc) causing Fusarium wilt in banana

Plant products along with biocontrol agents were tested against Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense (Foc). Of the 22 plant species tested, the leaf extract of Datura metel (10%) showed complete inhibition of the mycelial growth of Foc. Two botanical fungicides, Wanis 20 EC and Damet 50 EC along with selected PGPR strains with known biocontrol activity, Pseudomonas fluorescens 1, Pf1 and Bacillus subtilis, TRC 54 were tested individually and in combination for the management of Fusarium wilt under greenhouse and field conditions. Combined application of botanical formulation and biocontrol agents (Wanis 20 EC + Pf1 + TRC 54) reduced the wilt incidence significantly under greenhouse (64%) and field conditions (75%). Reduction in disease incidence was positively correlated with the induction of defense-related enzymes peroxidase (PO) and polyphenol oxidase (PPO). Three antifungal compounds (two glycosides and one ester) in D. metel were separated and identified using TLC, RP-HPLC (Reverse Phase-High Pressure Liquid Chromatography) and mass spectrometry. In this study it is clear that combined application of botanical formulations and biocontrol agents can be very effective in the management of Fusarium wilt of banana.     

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.     

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.     

Muscodor albus, a potential biocontrol agent against plant-parasitic nematodes of economically important vegetable crops in Washington State, USA

The fungus, Muscodor albus, was tested for nematicidal and nematostatic potential against four plant-parasitic nematode species with three different feeding modes on economically important vegetable crops in the Pacific Northwest. Meloidogyne chitwoodi, Meloidogyne hapla, Paratrichodorus allius, and Pratylenchus penetrans were exposed for 72 h to volatiles generated by M. albus cultured on rye grain in sealed chambers at 24 °C in the laboratory. In addition, the nematodes were inoculated into soil fumigated with M. albus, and incubated for 7 days prior to the introduction of host plants under greenhouse conditions. The mean percent mortality of nematodes exposed to M. albus in the chamber was 82.7% for P. allius, 82.1% for P. penetrans, and 95% for M. chitwoodi; mortality in the nontreated controls was 5.8%, 7%, and 3.9%, respectively. Only 21.6% of M. hapla juveniles died in comparison to 8.9% in controls. However, 69.5% of the treated juveniles displayed reduced motility and lower response to physical stimulus by probing, in comparison to the control juveniles. This is evidence of nematostasis due to M. albus exposure. The greenhouse study showed that M. albus caused significant reduction to all nematode species in host roots and in rhizosphere soil. The percent mortality caused by M. albus applied at 0.5% and 1.0% w/w in comparison to the controls was as follows: 91% and 100% for P. allius in the soil; 100% for P. penetrans in bean roots and soil; 85% and 95% for M. chitwoodi in potato roots, and 56% and 100% in the soil; 100% for M. hapla both in pepper roots and soil. In this study, M. albus has shown both nematostatic and nematicidal properties.     

Evaluation of formulations of Bacillus licheniformis for the biological control of tomato gray mold caused by Botrytis cinerea

Bacillus licheniformis N1, which has previously exhibited potential as a biological control agent, was investigated to develop a biofungicide to control the gray mold of tomato caused by Botrytis cinerea. Various formulations of B. licheniformis N1 were developed using fermentation cultures of the bacteria in Biji medium, and their ability to control gray mold on tomato plants was evaluated. The results of pot experiments led to the selection of the wettable powder formulation N1E, based on corn starch and olive oil, for evaluation of the disease control activity of this bacterium after both artificial infection of the pathogen and natural disease occurrence under production conditions. In plastic-house artificial infection experiments, a 100-fold diluted N1E treatment was found to be the optimum biofungicide spray formulation. This treatment resulted in the significant reduction of symptom development when N1E was applied before Bo. cinerea infection, but not after the infection. Both artificial infection experiments in a plastic house and natural infection experiments under production conditions revealed that the N1E significantly reduced disease severity on tomato plants and flowers. The disease control value of N1E on tomato plants was 90.5% under production conditions, as compared to the 77% conferred by a chemical fungicide, the mixture of carbendazim and diethofencarb (1:1). The prevention of flower infection by N1E resulted in increased numbers of tomato fruits on each plant. N1E treatment also had growth promotion activity, which showed the increased number of tomato fruits compared to fungicide treatment and non-treated control and the increased fruit size compared the non-treated control under production conditions. This study suggests that the corn starch-based formulation of B. licheniformis developed using liquid fermentation will be an effective tool in the biological control of tomato gray mold.     

一株防治香蕉枯萎病的短密木霉筛选及代谢物木霉素作用评价

由尖孢镰刀菌古巴专化型热带四号小种(Fusarium oxysporum f. sp. cubense tropical race4, FocTR4)引起的香蕉枯萎病(banana Fusarium wilt, BFW)是全世界范围内难以防治的真菌病害,给香蕉产业造成巨大的经济损失。本研究旨在筛选高效拮抗FocTR4的木霉生防菌株,并对其发酵代谢产物进行分离、提纯和鉴定,为香蕉枯萎病的高效生物防治提供重要生防菌株和活性化合物资源。从作物根际土壤中分离出木霉菌株,通过平板对峙培养、发酵液对病原菌孢子萌发及菌丝生长抑制,测试筛选出高效抑制FocTR4的生防木霉菌株;通过构建系统发育树明确生防菌株的分类地位;通过柱色谱法分离纯化菌株发酵液中活性成分,通过核磁共振波谱法(nuclear magnetic resonance spectroscopy, NMR)解析活性成分的结构;通过香蕉苗感病盆栽实验检测生防木霉菌株对香蕉枯萎病的防治效果。结果表明,本研究筛选到了1株拮抗FocTR4的菌株JSHA-CD-1003,平板对峙抑制率为60.6%;发酵液在24 h内能完全抑制FocTR4孢子萌发,7 d内对FocTR4菌丝生长的抑制率为52.6%;基于内转录间隔区(internal transcribed spacer, ITS)和tef1-α基因串联序列构建系统发育树,该菌株鉴定为短密木霉(Trichoderma brevicompactum),通过柱色谱法分离提纯和NMR鉴定单一活性化合物为木霉素(trichodermin),最小抑菌浓度(minimum inhibitory concentration, MIC)为25 μg/mL;盆栽生防实验表明,菌株JSHA-CD-1003发酵液对香蕉枯萎病的叶片黄化防治率为47.4%,球茎褐化防治率为52.0%。因此,JSHA-CD-1003通过产生木霉素有效抑制FocTR4孢子萌发和菌丝生长,对FocTR4引起的香蕉枯萎病具有良好的生物防治效果,是一株具有生防潜力的菌株。     

贝莱斯芽孢杆菌SH-1471发酵条件优化及其番茄枯萎病的防治效果

【目的】贝莱斯芽孢杆菌(Bacillus velezensis) SH-1471是一株兼具防控作物土传病害、促进土壤养分转化以及促进作物生长等功能的菌株(保藏编号：CCTCC No. M 2022923,专利号：ZL 2022 1 1479280.X)。挖掘其潜在的生物活性并探究其最适发酵条件,是推进该菌株产业化和商业化开发的有效手段之一。【方法】结合形态学、分子生物学、16SrRNA基因和gyrB基因对菌株SH-1471进行分类地位鉴定;利用PCR技术,对菌株抗生素合成基因检测;使用平板对峙试验和发酵液抑菌试验测定菌株抑菌广谱性;并测定其体外产酶、解磷、解钾、固氮及产铁载体能力;以菌株发酵液OD600值和抑菌率为指标,通过设计单因素试验和响应面优化试验,探究菌株的最佳发酵配方和最佳发酵条件;采用室内盆栽试验测定优化前后菌株发酵液对番茄植株的促生效果及其对番茄枯萎病的防治效果。【结果】经鉴定,SH-1471为贝莱斯芽孢杆菌(Bacillus velezensis),具有srfA、fenB、ituA、ituD和bymA等抗生素合成基因,对番茄枯萎病菌(Fusarium oxysporum...     

Elicitation of alkaloids in in vitro PLB (protocorm-like body) cultures of Pinellia ternata

The objective of this study was to determine the effect of two endophytic bacterial elicitors (Pseudomonas sp. and Enterobacter sp.) on the production of alkaloids in protocorm-like bodies (PLBs) of Pinellia ternata Breit. Both bacterial strains increased the growth rate of P. ternata PLBs. Pseudomonas sp. promoted the differentiation of the PLBs, whereas Enterobacter sp. inhibited PLB differentiation. The bacterial strains increased guanosine production in PLBs by 9–166%, inosine production by 2–33%, and trigonelline production by 114–1140% compared to the control. For Pseudomonas sp., guanosine and trigonelline production was greater when bacterial extracts were added to the PLB suspension cultures rather than living cells (co-culture treatment). Inosine production was similar in both the bacterial extract and co-culture treatments. For the Enterobacter sp., guanosine, inosine, and trigonelline production tended to be greatest when living cells were added to the PLB suspension cultures rather than bacterial extracts. These results suggest that Pseudomonas sp. and Enterobacter sp. could increase alkaloid yield from P. ternata under field or tissue culture conditions. We also observed that Pseudomonas sp. and Enterobacter sp. produced some of the same alkaloids as their host plants. Additional study needs to be done to determine if these endophytic bacteria could be used to produce alkaloids in the fermentation industry.     

Relationship between number and type of adhesions of Penicillium oxalicum conidia to tomato roots and biological control of tomato wilt

We studied (a) the extent adhesion of Penicillium oxalicum conidia to tomato roots after application of P. oxalicum conidial formulations with or without stickers, (b) the relationship between the extent of conidial adhesion to roots and biocontrol of the conidial formulations against tomato wilt, and (c) colonisation of roots by P. oxalicum. Adhesion of P. oxalicum conidia to tomato roots occurred within the first minute of contact between the root and the conidial formulation and the bonding strength was sufficiently strong to prevent conidial removal from the roots. In addition, some formulations with stickers that increased conidial adhesion to roots improved the biocontrol of tomato wilt, when compared to that of formulations without stickers. A “dried conidia without stickers” with 0.025% Nu-Film 17 had no effect on the biocontrol of tomato wilt, despite good adherence of the conidia to the roots. The numbers of P. oxalicum conidia that adhered to the roots was constant for 60 days after application of a “dried conidia without stickers” conidial formulation. The significance of these results (speed of adhesion, number of adhered conidia, and variability of conidial external surface) are discussed in relation to the biocontrol success of tomato wilt using different types of conidial formulations with and without stickers.     

番茄青枯病拮抗菌的定向筛选及其抗病促生机制研究

[目的] 从抑病型番茄根际土壤中筛选青枯病的高效拮抗促生菌,阐明其防病促生机制。[方法] 以番茄青枯雷尔氏菌（Ralstonia solanacearum）为靶病原菌,采用平板抑菌圈法,筛选拮抗菌;通过BOX-PCR指纹图谱鉴定菌株多样性,以平板透明圈法评价其产酶活性,并针对抑菌能力强、产酶种类多的拮抗菌开展16S rRNA基因系统发育分析;通过温室试验评价拮抗菌的防病促生能力,并在此基础上通过实时荧光定量PCR研究生防细菌对番茄青枯病的防病促生机制。[结果] 从番茄根际土壤分离获得29株细菌,其中15株对青枯菌具有拮抗功能,进一步通过BOX-PCR指纹图谱、酶活分析获得4株具有潜在防治番茄青枯病、促进生长的功能菌（B2、B5、B20、B23）,通过16S rRNA系统发育分析鉴定B2拮抗菌为解淀粉芽孢杆菌（Bacillus amyloliquefaciens）,B5和B20拮抗菌为枯草芽孢杆菌（Bacillus subtilis）,B23拮抗菌为贝莱斯芽孢杆菌（Bacillus velezensis）;温室试验表明,B2、B5、B20、B23拮抗菌的抑病效果分别为35.59%、8.47%、32.20%、96.61%,并且均能显著增加番茄生物量和生理性状,如地上部鲜重、总叶绿素含量、地下部根尖数等。B2、B5、B23拮抗菌显著促进番茄株高和根长,B2、B20、B23拮抗菌显著增加茎粗;而B23拮抗菌显著增加根系分叉数;实时荧光定量分析表明,B2、B20、B23拮抗菌株可促进抗病相关功能基因PR1α、POD1的表达量,B2、B5、B23拮抗菌促进吲哚乙酸（IAA）信号通路应答关键基因ctd1的表达量,B2、B5、B20、B23拮抗菌均降低乙烯（ETH）信号通路应答关键基因ERF2的表达量。[结论] 本研究分离筛选获得4株对番茄青枯病具有显著防治效果以及促进番茄生长的PGPR菌株,可为定向筛选植物促生防病菌提供理论依据。     

Evaluation of Beauveria bassiana and B. brongniartii strains and four wild-type fungal species against adults of Bactrocera oleae and Ceratitis capitata

The virulence of two isolates of the hyphomycete fungi, Beauveria bassianaand B. brongniartii, and additional fungal species isolated from diseased Bactrocera oleae pupae and Sesamia nonagrioideslarvae were assessed against adults of the olive fruit fly B. oleae and the Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae). Contact and oral bioassays revealed that moderate to high mortality rates for the olive fruit fly occurred when the adults were exposed to conidia of Mucor hiemalis, Penicillium aurantiogriseum, P. chrysogenum and B. bassianaisolates. A strain of M. hiemalis isolated from S. nonagrioides larvae was the most toxic resulting in 85.2% mortality to the olive fruit fly adults. B. brongniartiiand B. bassiana were the most pathogenic to the C. capitataadults causing 97.4 and 85.6% mortality. Metabolites collected from the M. hiemalis and P. chrysogenum isolates were toxic to adults of both species.     

Zn(II)对好氧反硝化菌Acinetobacter sp. JR-142的代谢活性影响

【目的】研究不同Zn(Ⅱ)浓度对好氧反硝化菌Acinetobacter sp.JR-142生理活性,尤其是反硝化代谢特性的影响。【方法】筛选一株好氧反硝化菌,优化了最佳活性条件;分析了不同Zn(Ⅱ)浓度对生长曲线和反硝化效率的影响以及对细胞形态的影响;明晰了不同Zn(Ⅱ)浓度条件下,细胞特征活性酶-硝酸盐还原酶和亚硝酸盐还原酶的活性变化情况,分析了不同活性同关键酶的编码基因napA和nirS的相对表达量之间的规律。【结果】获得一株具有好氧反硝化功能的菌株,命名为JR-142,经鉴定为不动杆菌Acinetobacter sp.。在以琥珀酸钠为碳源,C/N为6,pH为7.0,温度30℃,转速为180 r/min的条件下,好氧反硝化活性最高。结果表明当Zn(Ⅱ)浓度为3.25 mg/L时,对菌株的生长及好氧反硝化速率有促进作用;当Zn(Ⅱ)浓度为52 mg/L以上浓度时,菌株的生长及反硝化速率均受到抑制。酶活及关键基因napA、nirS的定量分析结果显示,对照组及JR+0.05处理组的硝酸盐还原酶NR、亚硝酸盐还原酶NiR活性均高于JR+0.8处理组,在24 h时,JR+0.05 Zn(Ⅱ)处理组中,细胞的关键好氧反硝化基因napA及nirS的相对表达量显著高于对照组,这进一步说明3.25 mg/L Zn(Ⅱ)可以促进好氧反硝化过程,而在24 h及32 h时对照组及JR+0.05处理组的基因相对表达量远高于JR+0.8处理组,也说明52 mg/L Zn(Ⅱ)则会对反应产生抑制。【结论】探究并系统分析了不同Zn(Ⅱ)浓度对不动杆菌Acinetobacter sp.JR-142生长繁殖以及和在重金属锌离子存在的情况下影响好氧反硝化生理活性的影响,为后续硝酸盐-重金属复合污染废水的生物处理技术提供了数据指导。