Bacillus sp. L324-92 for Biological Control of Three Root Diseases of Wheat Grown with Reduced Tillage

ABSTRACT Strain L324-92 is a novel Bacillus sp. with biological activity against three root diseases of wheat, namely take-all caused by Gaeumannomyces graminis var. tritici, Rhizoctonia root rot caused by Rhizoctonia solani AG8, and Pythium root rot caused mainly by Pythium irregulare and P. ultimum, that exhibits broad-spectrum inhibitory activity and grows at temperatures from 4 to 40 degrees C. These three root diseases are major yieldlimiting factors for wheat in the U.S. Inland Pacific Northwest, especially wheat direct-drilled into the residue of a previous cereal crop. Strain L324-92 was selected from among approximately 2,000 rhizosphere/rhizoplane isolates of Bacillus species isolated from roots of wheat collected from two eastern Washington wheat fields that had long histories of wheat. Roots were washed, heat-treated (80 degrees C for 30 min), macerated, and dilution-plated on (1)/(10)-strength tryptic soy agar. Strain L324-92 inhibited all isolates of G. graminis var. tritici, Rhizoctonia species and anastomosis groups, and Pythium species tested on agar at 15 degrees C; provided significant suppression of all three root diseases at 15 degrees C in growth chamber assays; controlled either Rhizoctonia root rot, takeall, or both; and increased yields in field tests in which one or more of the three root diseases of wheats were yield-limiting factors. The ability of L324-92 to grow at 4 degrees C probably contributes to its biocontrol activity on direct-drilled winter and spring wheat because, under Inland Northwest conditions, leaving harvest residues of the previous crop on the soil surface keeps soils cooler compared with tilled soils. These results suggest that Bacillus species with desired traits for biological control of wheat root diseases are present within the community of wheat rhizosphere microorganisms and can be recovered by protocols developed earlier for isolation of fluorescent Pseudomonas species effective against take-all.     

Effect of Population Density of Pseudomonas fluorescens on Production of 2,4-Diacetylphloroglucinol in the Rhizosphere of Wheat

ABSTRACT The role of antibiotics in biological control of soilborne pathogens, and more generally in microbial antagonism in natural disease-suppressive soils, often has been questioned because of the indirect nature of the supporting evidence. In this study, a protocol for high pressure liquid chromatography/mass spectrometry is described that allowed specific identification and quantitation of the antibiotic 2,4-diacetylphloroglucinol (Phl) produced by naturally occurring fluorescent Pseudomonas spp. on roots of wheat grown in a soil suppressive to take-all of wheat. These results provide, for the first time, biochemical support for the conclusion of previous work that Phl-producing fluorescent Pseudomonas spp. are key components of the natural biological control that operates in take-all-suppressive soils in Washington State. This study also demonstrates that the total amount of Phl produced on roots of wheat by P. fluorescens strain Q2-87, at densities ranging from approximately 10(5) to 10(7) CFU/g of root, is proportional to its rhizosphere population density and that Phl production per population unit is a constant (0.62 ng/10(5) CFU). Thus, Phl production in the rhizosphere of wheat is strongly related to the ability of the introduced strain to colonize the roots.     

Interactions Between Strains of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens in the Rhizosphere of Wheat

ABSTRACT Strains of fluorescent Pseudomonas spp. that produce the antibiotic 2,4-diacetylphoroglucinol (2,4-DAPG) are among the most effective rhizobacteria controlling diseases caused by soilborne pathogens. The genotypic diversity that exists among 2,4-DAPG producers can be exploited to improve rhizosphere competence and biocontrol activity. Knowing that D-genotype 2,4-DAPG-producing strains are enriched in some take-all decline soils and that P. fluorescens Q8r1-96, a representative D-genotype strain, as defined by whole-cell repetitive sequence-based polymerase chain reaction (rep-PCR) with the BOXA1R primer, is a superior colonizer of wheat roots, we analyzed whether the exceptional rhizosphere competence of strain Q8r1-96 on wheat is characteristic of other D-genotype isolates. The rhizosphere population densities of four D-genotype strains and a K-genotype strain introduced individually into the soil were significantly greater than the densities of four strains belonging to other genotypes (A, B, and L) and remained above log 6.8 CFU/g of root over a 30-week cycling experiment in which wheat was grown for 10 successive cycles of 3 weeks each. We also explored the competitive interactions between strains of different genotypes inhabiting the same soil or rhizosphere when coinoculated into the soil. Strain Q8r1-96 became dominant in the rhizosphere and in nonrhizosphere soil during a 15-week cycling experiment when mixed in a 1:1 ratio with either strain Pf-5 (A genotype), Q2-87 (B genotype), or 1M1-96 (L genotype). Furthermore, the use of the de Wit replacement series demonstrated a competitive disadvantage for strain Q2-87 or strong antagonism by strain Q8r1-96 against Q2-87 in the wheat rhizosphere. Amplified rDNA restriction analysis and sequence analysis of 16S rDNA showed that species of Arthrobacter, Chryseobacterium, Flavobacterium, Massilia, Microbacterium, and Ralstonia also were enriched in culturable populations from the rhizosphere of wheat at the end of a 30-week cycling experiment in the presence of 2,4-DAPG producers. Identifying the interactions among 2,4-DAPG producers and with other indigenous bacteria in the wheat rhizosphere will help to elucidate the variability in biocontrol efficacy of introduced 2,4-DAPG producers and fluctuations in the robustness of take-all suppressive soils.     

生防菌与茄病镰刀菌在黄瓜根际动态变化

应用荧光定量PCR和稀释分离法检测了枯草芽孢杆菌Bacillus subtilis和木霉菌Trichoderma spp.与茄病镰刀菌Fusariumsolani f.sp.cucurbitae在黄瓜根际动态变化及其对黄瓜根腐病的防治效果。结果表明,施用500×木霉菌肥、100×枯草芽孢杆菌7d后对黄瓜根腐病的防效分别为100%和92.5%,14和28d的防效分别为96.71%、86.76%和85.35%、79.24%;100×枯草芽孢杆菌初始拷贝数为130787 copys/μL,7d内枯草芽孢杆菌拷贝数下降到51161 copys/μL,14和28d时数量开始上升到295139和680556 copys/μL。木霉菌肥的变化趋势与枯草芽孢杆菌相同;用枯草芽孢杆菌和木霉菌肥防治黄瓜根腐病后,茄病镰刀菌变化趋势一致,初始拷贝数分别为2.61和15.34 copys/μL,7d内茄病镰刀菌DNA拷贝数增加明显,100×枯草芽孢杆菌和木霉菌DNA拷贝数分别为11.22和20.9 8copys/μL,之后茄病镰刀菌数量保持平稳。经过稀释分离法分离2种生防菌和茄病镰刀菌的数量,变化趋势与荧光定量PCR检测趋势相似。因此使生防菌快速定殖是提高其对土传病害防治效果的重要因素之一。     

Fusarium oxysporum f. sp. lycopersici retardation through induction of defensive response in tomato plants using a liquid formulation of Pseudomonas fluorescens (Pf1)

A liquid based Pseudomonas fluorescens (Pf1) bioformulation was found to contribute the restriction of Fusarium oxysporum f. sp. lycopersici in tomato roots by inducing defence enzymes. Induction of defence enzymes such as phenylalanine ammonia lyase (PAL), peroxidase (PO), polyphenoloxidase (PPO), catalase, β-1,3 glucanase and super oxide dismutase (SOD), was studied in tomato plants pretreated with liquid as well as a talc based formulation of Pf1 challenged with F. oxysporum f. sp. lycopersici in glasshouse vegetable production systems. There were increased activities of PAL, PO, PPO, catalase and β-1 3-glucanases in tomato plants treated with a combined application of seedling dip?+?soil application?+?foliar spray of liquid and talc formulation of Pf1 when compared to pathogen inoculated and untreated healthy controls. The activities of the above enzymes started to increase at 3rd day, reached maximum levels on 8-9th day and thereafter declined gradually. Similarly, native polyacralamide gel electrophoresis (PAGE) analysis revealed that one to six isoforms of the defence enzymes each with a higher intensity were expressed in these treatments, whereas fewer isoforms with less intensity were noticed in inoculated controls. These results suggest that the retardation of the invasion of F. oxysporum f. sp. lycopersici in tomato roots resulting from treatment with the liquid formulation of Pf1 was due to enhancement of activities of enzymes involved in the phenylpropanoid pathway. These results suggest that induced systemic resistance occurred in the treated tomato plants.     

内生菌荧光假单胞菌DLJ1和蜡状芽胞杆菌SZ5对南方根结线虫胁迫下辣椒植株抗性与产量品质的影响

为了检验内生菌荧光假单胞菌DLJ1和蜡状芽胞杆菌SZ5的促生与提高植株对南方根结线虫的防治效果, 确定合适的菌剂接种方式, 并分析其机制, 本文以杀线剂阿维菌素为对照, 比较了不同接菌方式下DLJ1 和SZ5对辣椒植株在南方根结线虫胁迫下的抗性以及对产量品质的影响?结果证实DLJ1在种子萌发期的一次接菌与萌发期与田间的5次接菌处理均可使根系根结数减少70%以上, 使植株对南方根结线虫的抗性升至高抗级别, 辣椒产量提高超过150%; DLJ1一次接菌处理组的果型最大?Vc与可溶性蛋白含量最高?SZ5萌发期一次处理根结数降低40%, 植株对南方根结线虫表现为中抗, 产量提高60%以上; SZ5多次接菌处理根结数只能降低27%, 产量仅提高6%左右?阿维菌素处理的效果介于DLJ1和SZ5之间, 可降低植株根结数45%, 提高产量49%左右?分析其机理发现, 两菌剂处理均可通过提高植株的POD酶活性和总酚含量, 降低O2-产生速率和MDA积累, 提高光合色素含量等来促进植物生长, 通过提高几丁质酶活性来提高植株对线虫的抗性?总之, 只需要在辣椒种子萌发期进行一次DLJ1接菌处理, 即可使植株获得稳定而显著的促生和防治南方根结线虫双重效果, 提示DLJ1菌在农业生产上的应用前景广阔?     

栎空腔瘿蜂及其天敌长尾小蜂种群数量消长动态

栎空腔瘿蜂Trichagalma acutissimae(Monzen)是危害栓皮栎Quercus variabilis Blume叶片的重要林业害虫,为我国新纪录种。近年来,在南太行山区的栓皮栎林内暴发成灾,长尾小蜂Torymus sp.是栎空腔瘿蜂的一种主要寄生性天敌,为明确栎空腔瘿蜂及其天敌长尾小蜂种群数量消长动态,为预测预报和综合治理提供依据,本文调查了栎空腔瘿蜂和长尾小蜂成虫种群动态以及长尾小蜂的寄生率。结果表明,长尾小蜂成虫种群数量及寄生率一年发生2个高峰,第1个高峰发生在5月中旬,第2个高峰发生在7月下旬;2011—2015年,无性世代虫瘿平均每株发生的数量分别为826.0、816.4、61.8、11.0和3.4个,平均每块粘虫板长尾小蜂成虫发生数量分别为39.00、46.33、19.30、13.33和8.30头,长尾小蜂总寄生率分别为39.7%、51.0%、61.1%、54.9%和29.5%。     

Population Dynamics of Fusarium spp. and Microdochium nivale in Crops and Crop Residues of Winter Wheat

ABSTRACT Naturally occurring populations of Fusarium avenaceum, F. culmorum, F. graminearum, F. poae, and Microdochium nivale were studied in two field experiments from anthesis in June 2003 until harvest in crops of winter wheat, and subsequently during 10 months after harvest until June 2004 on their residues exposed on the soil surface under field conditions. The dynamics of the different pathogens were estimated by quantifying the amount of DNA present in wheat tissues using TaqMan-polymerase chain reaction. While colonization of grain by Fusarium spp. and M. nivale was low, high amounts of DNA of F. avenaceum, F. graminearum, and F. culmorum were found in ear residues, internodes, and nodes of the mature crop. Amounts of DNA of pathogens decreased significantly during the following 10 months in residues of internodes and nodes, but not in residues of stem bases. Knowledge on population dynamics of pathogens will help to develop preventive measures aimed at reduction of inoculum sources of head blight pathogens.     

假单胞茵YT3和枯草芽孢杆菌DZl的发酵罐生长条件优化

假单胞菌（Pseudomonas sp．）YT3和枯草芽孢杆菌（Bacillus subtilis）DZl是从植物根际土壤筛选出的具有应用潜力的植物根际促生细菌。研究发酵罐中接种量、转速、通气量等因素对YT3菌量以及对DZl芽孢率、菌量的影响。结果表明：不同的接种量、转速和通气量对Pseudomonas sp．YT3的活菌数、Bacillussubtilis DZl的活菌数及芽孢率均有显著影响。10％的接种量、2m3／ll的通气量对YT3和DZl菌株的作用效果与15％的接种量、3m3／h相当,但显著高于5％的接种量和1m3／h。当转速为200r／min时,YT3具有最高的活菌数,DZl具有最高的活菌数和芽孢率。综合分析认为,YT3和DZl发酵罐的最佳条件为接种量10％、转速200r／min和通气量2矗m。     

甘肃甘谷县麦蚜种群动态及防治指标初探

本文通过1991～1994年甘谷县麦蚜田间系统调查和大田普查，明确了麦蚜的种群结构和消长动态及天敌与麦蚜的跟随关系。并建立了麦蚜混合种群数量与产量损失率的回归模型即Y＝0.3591+0.001897x，依次确定了小麦穗期混合麦蚜种群的防治指标为百株蚜量1000头。     

Biological control of Ralstonia wilt,Phytophthora blight,Meloidogyne root-knot on bell pepper by the combination of Bacillus subtilis AR12, Bacillus subtilis SM21 and Chryseobacterium sp. R89

In a previous study, we screened the combination of the three bacterial strains—Bacillus subtilis AR12, Bacillus subtilis SM21, and Chryseobacterium sp. R89—(BBC), which can control mixed diseases, including Ralstonia wilt, Phytophthora blight, and Meloidogyne root-knot on bell pepper with high biocontrol efficacy and yield increase under greenhouse and field conditions. In this study, to achieve the best biocontrol agents for separately controlling Ralstonia wilt, Phytophthora blight and Meloidogyne root-knot by BBC, the biocontrol efficacy by BBC-related BCAs (AR12, SM21, R89, AR12 + SM21, AR12 + R89, SM21 + R89 and AR12 + SM21 + R89) were compared regarding all three single diseases and the mixed diseases, respectively. BBC achieved the highest biocontrol efficacy against all three single diseases and the mixed diseases (biocontrol efficacies of 92.46 %, 81.81 %, 86.49 % and 87.31 % against Ralstonia wilt, Phytophthora blight, Meloidogyne root-knot and the mixed diseases, respectively), which were significantly more than that attained by the three single strains and the two-strain combinations AR12 + SM21, AR12 + R89 and SM21 + R89. Although a biomass increase of 4.86–21.14 % was attained by BBC-related BCAs without BBC, BBC achieved a maximum biomass increase of 64.67 %.     

Dominant Colonisation of Wheat Roots by Pseudomonas fluorescens Pf29A and Selection of the Indigenous Microflora in the Presence of the Take-all Fungus

Increases in populations of fluorescent pseudomonads on wheat roots are usually associated with take-all decline, natural control of take-all, a disease caused by the fungus Gaeumannomyces graminis var. tritici (Ggt). Colonisation by Pseudomonas fluorescens strain Pf29A was assessed on the roots of healthy plants and of plants with take-all, and the effect of this bacterium on indigenous populations of fluorescent pseudomonads was studied. The efficacy of Pf29A as an agent for the biocontrol of take-all on five-week-old wheat seedlings was tested in non-sterile conducive soil in a growth chamber. RAPD (random amplification of polymorphic DNA) fingerprinting with a decamer primer was used to monitor strain Pf29A and culturable indigenous rhizoplane populations of fluorescent pseudomonad. Pf29A decreased disease severity and accounted for 44.6% of the culturable fluorescent pseudomonads on healthy plant rhizoplane and 75.8% on diseased plant rhizoplane. Fewer RAPD patterns were obtained when Pf29A was introduced into the soil with Ggt. In the presence of Ggt and necrotic roots, Pf29A became the dominant root coloniser and dramatically changed the diversity and the structure of indigenous fluorescent pseudomonad populations. The results show that Ggt and reduced lesion size on roots can trigger a specific increase in antagonist populations and that the introduction of a biocontrol agent in soil influences the structure of indigenous bacterial populations.     

杨始叶螨发生规律研究

杨始叶螨以受精雌成螨在柳树的树干裂缝内、翘皮下、虫孔内越冬。翌年３月底日均温达７℃以上，柳树展叶盛期开始出蜇，４月上旬为出蜇盛期（占出蜇总量的８６．９％）。全年发生盛期在５月中旬至７月上旬。该螨成螨喜在卷叶、叠叶、瘤瘿叶内群集、吐丝拉网。根据该螨的发育起点温度和有效积温，利用泰安市气象局温度资料，计算出该螨在泰安地区１ａ可发生１１代，最少１０代，最多１２代     

Use of a Nitrate-Nonutilizing Mutant and Selective Media to Examine Population Dynamics of Fusarium oxysporum f. sp. spinaciae in Soil

ABSTRACT Determining the population density of the spinach wilt pathogen Fusarium oxysporum f. sp. spinaciae in soil with conventional Fusarium-selective media is quite difficult because nonpathogenic strains of F. oxysporum also grow on those media and are indistinguishable from the pathogen. Therefore, a nitrate-nonutilizing (nit) mutant of the pathogen and corresponding selective media were tested in an experimental approach to determine the population density of the pathogen. Colony forming units of the pathogen were countable after soil-dilution plating onto nit mutant-selective media MMCPA, CMP, and CGMBP. Colony forming units of wild-type Fusarium spp. were countable using a wildtype Fusarium-selective medium, GMBP. By combining nit mutant- and wild-type-selective media, the population densities of pathogenic and nonpathogenic F. oxysporum in the same soil could be measured selectively. This method was useful in studying population dynamics of the pathogen after different soil treatments. Soil disinfested with hot water or chloropicrin was amended with the nit mutant pathogen, and subsequent changes in population densities of the pathogen were compared with those in nontreated field soil. The pathogen rapidly proliferated in disinfested soil and wilt developed faster than in nontreated soil. When a nonpathogenic isolate of F. oxysporum was added at high density to sterilized soil prior to the pathogen, growth of the pathogen was greatly suppressed. Nonpathogenic F. oxysporum could not, however, reduce the density of preexisting pathogen.     

Effect of Bacillus thuringiensis subsp. israelensis and neonicotinoid insecticides on the fungus gnat Bradysia sp nr. coprophila (Lintner) (Diptera: Sciaridae)

The soil bacterium Bacillus thuringiensis Berliner subsp. israelensis (Bti), the neonicotinoid insecticides dinotefuran, imidacloprid, thiamethoxam and clothianidin and the insect growth regulator pyriproxyfen were evaluated to determine their efficacy against the larval stages of the fungus gnat Bradysia sp nr. coprophila (Lintner) in the laboratory. Treatments were applied as a drench to the growing medium in polypropylene deli containers. The Bti treatments had no effect on either instar tested, whereas all the other compounds negatively affected both the second and third instars. This study demonstrates that the soil bacterium B. thuringiensis var. israelensis may not be active on these larval stages, whereas the neonicotinoid insecticides and the insect growth regulator pyriproxyfen are effective on these stages. The fact that Bti is not effective on the second and third instars of the fungus gnat means that greenhouse producers using this insecticide must make applications before fungus gnat populations build up and before overlapping generations develop.     

Development and evaluation of water-in-oil formulation of Pseudomonas fluorescens (FP7) against Colletotrichum musae incitant of anthracnose disease in banana

Banana anthracnose incited by Colletotrichum musae (Berk & Curt.) Arx. is a serious disease both in the field and after harvest. Nineteen isolates of antagonistic bacteria and twelve isolates of yeast were screened for the biocontrol activity against this disease. Pseudomonas fluorescens (FP7) had the highest inhibition rate of mycelial growth of C. musae. Water-in-oil formulation of P. fluorescens (FP7) was developed by adding various oils such as coconut (28.50 %), rice bran (28.50 %) and castor (28.50 %) individually to the bacterial culture. Addition of these oils allowed bacterial populations to survive at various levels up to 210 days of storage, the duration of experiment; whereas in the control treatment (NA broth) it survived at high levels only up to 45 days. Biochemical and antibiotic characterization of P. fluorescens (FP7) in water in oil formulation showed more intensity up to 180 days. Two field trials were conducted to standardize the water in oil formulation. The application of water-in-oil formulation significantly increased the yield (63.56 t/ha), and nutritional factors.     

Effects of ametryn [2-(ethylamino)-4-(isopropylamino)-6-(methylthio)-s-triazine] on nitrate reductase activity and nitrite content of wheat (Triticum aestivum L.)

The objectives of this study were to show that: (a) a herbicide, such as ametryn, which interferes with the photosynthetic electron transport system, causes nitrite to accumulate in illuminated leaves and (b) that nitrite is toxic and contributes to the herbicidal damage and death of the plant. Tests were conducted on wheat seedlings grown on 5 mM nitrate, 5 mM ammonia, and zero nitrogen. Ametryn treatment decreased in vivo and in vitro nitrate reductase activity (NRA) within a 26-hr period. In vivo NRA decreased more rapidly than in vitro NRA. Compared with control tissue, only 3% in vivo NRA remained at the end of 26 hr. The in vivo assay conducted in light confirmed the inhibition of photosynthetic electron flow by ametryn within the leaf tissue. Nitrate-grown, ametryn-treated plants accumulated nitrite and, after 10 days were the only plants that were completely desiccated and dead. Ammonia- and zero-nitrogen, ametryn-treated plants did not accumulate nitrite, were only partially chlorotic after the 10-day period, and were still living. Low levels of NO_(X) (NO₂ and/or NO) emissions were demonstrated by nitrate-grown ametryn-treated plants.     

Aerial Application of Bacillus thuringiensis (Dipel) against Colotois (Himera) pennaria L. in Comparison with ULV-Application of Various Insecticides 1)

In spring 1972 experiments were carried out in Lower Austria with the Bacillus thuringiensis isolate Dipel in order to assess its effectiveness in controlling Colotois pennaria L. infesting oak and other broad-leaved trees. The results obtained compared favourably with those produced by the insecticides Nexion²), malathion and Dipterex³), although both malathion and Dipterex yielded higher mortality. Similar results were obtained when B. thuringiensis was used to control Stilpnotia salicis L., feeding on poplar trees. Neodiprion sertifer Geoffr. and Pris-tiphora abietina Christ., however, were not affected.     

Cytological Evidence of an Active Role of Silicon in Wheat Resistance to Powdery Mildew (Blumeria graminis f. sp. tritici)

ABSTRACT Silicon (Si) amendments in the form of exogenously supplied nutrient solution or calcium silicate slag protect wheat plants from powdery mildew disease caused by the fungus Blumeria graminis f. sp. tritici. The most striking difference between Si- and Si+ plants challenged with B. graminis f. sp. tritici was the extent of epidermal cell infection and colonization by B. graminis f. sp. tritici. Histological and ultrastructural analyses revealed that epidermal cells of Si+ plants reacted to B. graminis f. sp. tritici attack with specific defense reactions including papilla formation, production of callose, and release of electron-dense osmiophilic material identified by cytochemical labeling as glycosilated phenolics. Phenolic material not only accumulated along the cell wall but also was associated with altered integrity of haustoria in a manner similar to localized phytoalexins as reported from other pathosystems. These results strongly suggest that Si mediates active localized cell defenses against B. graminis f. sp. tritici attack.