Biological control of onion leaf blight disease by bulb and foliar application of powder formulation of antagonist mixture

Abstract

Three antagonists: Pseudomonas fluorescens (Pf1), Bacillus subtilis and Trichoderma viride, were tested alone and in combination for suppression of onion leaf blight (Alternaria palandui) disease under glasshouse and field conditions. The average mean of disease reduction was 24.81% for single strains and 42.44% for mixtures. In addition to disease suppression, treatment with a mixture of antagonists promoted plant growth in terms of increased plant height and ultimately bulb yield. Though seed treatment of either single strain or strain mixtures alone could reduce the disease, subsequent application to root, leaves or soil further reduced the disease and enhanced the plant growth. The mixture consisting of Pseudomonas fluorescens Pf1 plus Bacillus subtilis plus Trichoderma viride was the most effective in reducing the disease and in promoting plant growth and bulb yield in greenhouse and field tests.     

Biological control of bacterial wilt caused by Pseudomonas solanacearum in India with antagonistic bacteria

From among 125 strains of fluorescent and 52 strains of nonfluorescent bacteria initially screened in the laboratory for their antibiosis towards the bacterial wilt pathogen, Pseudomonas solanacearum, strain Pfcp of Pseudomonas fluorescens and strains B33 and B36 of Bacillus spp., were chosen and evaluated further in greenhouse and field tests. Pfcp treated banana (Musa balbisiana), eggplant and tomato plants were protected from wilt upto 50, 61 and 95% in greenhouse and upto 50, 49 and 36% respectively in field. Protection afforded by the Bacillus strains was lower. In bacteria-treated plants which were subsequently inoculated with P. solanacearum plant height and biomass values increased and were close to those of nontreated and noninoculated control plants.     

Antagonistic and antimicrobial activities of some bacterial isolates collected from soil samples

Thirty seven bacterial cultures isolated from soil samples obtained from different locations were tested for their antagonistic activity against some fungal pathogens, viz., Sclerotium rolfsii, Fusarium oxysporum and Rhizoctonia solani, causal agents of collar rot of sunflower, wilts and root rots, respectively. Among them, 5 bacterial strains, viz., A1 6 (Bacillus sphaericus), K1 24 (Pseudomonas fluorescens), M1 42 (Bacillus circulans), M1 66 (Bacillus brevis) and T1 22 (Bacillus brevis) showed positive antagonistic activity. M1 66 was the most effective in inhibiting mycelial growth of S. rolfsii in vitro followed by M1 42, T1 22, K1 24 and A1 6. Only one bacterial strain i.e. M1 42 exhibited antagonistic activity against F. oxysporum, and none of the bacterial strains gave positive activity against R. solani. Furthermore, antimicrobial activities of all the 5 strains were checked against different test organisms. These strains showed their extensive inhibition effect particularly against gram-positive test bacteria (Staphylococcus aureus and Bacillus subtilis) and the test fungal strain (Candida albicans). On the other hand, B. brevis M1 66 and B. brevis T1 22 strains had an inhibitory effect against gram positive and gram-negative test bacteria (Escherichia coli and Proteus vulgaris) as well as the test fungal strain.     

Evaluation of indigenous bacterial strains for biocontrol of the frogeye leaf spot of soya bean caused by Cercospora sojina

Aims: Assessment of biological control of Cercospora sojina, causal agent of frogeye leaf spot (FLS) of soya bean, using three indigenous bacterial strains, BNM297 (Pseudomonas fluorescens), BNM340 and BNM122 (Bacillus amyloliquefaciens). Methods and Results: From cultures of each bacterial strain, cell suspensions and cell‐free supernatants were obtained and assayed to determine their antifungal activity against C. sojina. Both mycelial growth and spore germination in vitro were more strongly inhibited by bacterial cell suspensions than by cell‐free supernatants. The Bacillus strains BNM122 and BNM340 inhibited the fungal growth to a similar degree (I～52–53%), while cells from P. fluorescens BNM297 caused a lesser reduction (I～32–34%) in the fungus colony diameter. The foliar application of the two Bacillus strains on soya bean seedlings, under greenhouse conditions, significantly reduced the disease severity with respect to control soya bean seedlings and those sprayed with BNM297. This last bacterial strain was not effective in controlling FLS in vivo. Conclusions: Our data demonstrate that the application of antagonistic bacteria may be a promising and environmentally friendly alternative to control the FLS of soya bean. Significance and Impact of the Study: To our knowledge, this is the first report of biological control of C. sojina by using native Bacillus strains.     

Segregation pattern of gene expression in cotton leaf curl virus-resistant transgenics

Non-pathogenic soil bacteria living in association with roots of higher plants enhance their adaptive potential and thus could be beneficial for their growth. Here, we present the current status of the use of Bacillus subtilis in biocontrol. Rhizobacteria are found in the rhizosphere. Plant growth promoting rhizobacteria (PGPR) strains, such as Bacillus and Pseudomonas, were isolated by using Nutreint dextrose Agar medium or Potato Dextrose Agar medium. The selection of PGPR strains was done by duel culture methods against the potato pathogens. The interaction of PGPR (Bacillus) with potato seeds or vegetative parts show promising antagonism by virtue of producing siderophore and antibiotics against black scurf and stem canker diseases of potato caused by Rhizoctonia solani, thereby resulting in increase of potato yield. The effectiveness of PGPR strain (Bacillus spp.) in improving the yield of potato in greenhouse conditions and in the field was observed.     

Evaluation of bacterial antagonists of Ralstonia solanacearum,causal agent of bacterial wilt of potato

Bacterial wilt of potato caused by Ralstonia solanacearum is one of the most destructive diseases in Kurdistan province, Iran. The objective of the present study was to evaluate antagonistic effects of some rhizobacteria isolated from the rhizosphere of potato plants against R. solanacearum, the agent of potato bacterial wilt. A total of 52 rhizobacteria were isolated and screened for in vitro antagonistic activity against R. solanacearum. Seven isolates with inhibiting effects of the pathogen were identified by phenotypic properties and partial sequencing of 16s rRNA as Pseudomonas fluorescens Pf11, P. fluorescens Pf16, Pseudomonas putida Pp17, Paenibacillus sp. Pb28 and Enterobacter sp. En38, Pseudomonas fluorescens Pp23 and Serratia sp. Se40. Strains Pf11, Pf16, Pp17 and Pb28 significantly inhibited the growth of the pathogen. Strains En38, Pp23 and Se40 showed a moderate or weak inhibition. During greenhouse study, strains were evaluated for their effects in reducing of disease and increasing biomass of potato plants. In according to greenhouse experiment results, isolates Pb28, Pp17 and Pf11significantly reduced disease by 55.56%, 51.50% and 38.58%, respectively. In addition, plant biomass significantly increased in plants treated with Pb28, Pp17, Pf11 and Pf16, compared to the control. Therefore, this study shows that these four strains have potential to be used as biocontrol agents against R. solanacearum. To confirm their effectiveness as commercial biocontrol agent, it is necessary to evaluate their efficiency in the field conditions in the next studies.     

丁香内生死亡谷芽孢杆菌DX78的分离鉴定及其抑菌成分

【背景】植物内生菌往往产生与植物相同、相似或新颖的次生代谢产物,丁香具有广谱优异的抗菌活性,可从中分离到强抑菌作用的内生细菌。【目的】筛选抑制姜瘟病菌的丁香内生细菌并分离其活性成分。【方法】牛津杯法筛选拮抗内生细菌;根据16S rRNA基因序列鉴定菌株;有机溶剂萃取、硅胶柱层析和薄层制备色谱分离活性成分;测定1HNMR、13CNMR和DEPT(135°)并对分离的活性成分进行结构鉴定;滤纸片法和菌丝生长速率法测定活性成分抑菌活性。【结果】共分离到112株丁香内生细菌,从叶中分离最多,占37.4%。其中17株对姜瘟病菌有抑制,DX78菌株抑制作用最好,经鉴定为死亡谷芽孢杆菌,并从其发酵液中追踪分离到邻苯二甲酸二丁酯(dibutyl phthalate, DBP)。DBP对姜瘟病菌、猕猴桃溃疡病菌的MIC分别为0.3 mg/disc、0.25 mg/disc;其还可抑制多种植物病原真菌,特别对苹果炭疽叶枯病菌、番茄灰霉病菌和苹果腐烂病菌的抑制EC50仅分别为3.751、18.568和22.019μg/mL。以苹果炭疽叶枯病菌为靶标菌,戊唑醇抑制毒力约为DBP抑制毒力的4.5倍,DBP抑制毒...     

Screening and characterization of antifungal clusterbean (Cyamopsis tetragonoloba) rhizobacteria

About 377 guar (Cyamopsis tetragonoloba) rhizobacteria were isolated from cultivated soils of north-west India (Thar Desert) and their antifungal activity against Macrophomina phaseolina (strains of groundnut, mungbean and guar) and Fusarium oxysporum (strains of chickpea and cumin) was examined. Isolates were characterised for generic types and physiological/functional diversity. About 19% isolates representing 24% locations were inhibitory to fungal growth. Isolates 009071, 009073, 009078 and 102354 recorded maximum inhibition of pathogenic fungi on plates. Isolate 034206 gave highest %RI, 009073 showed maximum protease activity and 102354 gave highest salt tolerance. Net house and field screening results revealed that isolates 004052, 009071, 009073, 001001, 094340 and 102354 had potential for biocontrol of disease. Partial sequencing of 16S rRNA gene of 61 isolates showed that 85% of isolates belonged to genus Bacillus. Phylogenetically, however, there were four clusters in the Bacillus group comprising of Bacillus subtilis, B. cereus, B. pumilus and B. sphaericus. One isolate was identified as B. flexus, while six isolates were Bacillus spp. Four isolates were identified as Achromobacter xylosoxidans, two as Bacterium (unclassified bacteria), and one each as Ochrobactrum intermedium, Pseudomonas aeruginosa and Ralstonia sp.     

Protection of canola (Brassica napus) against fungal pathogens by strains of biocontrol rhizobacteria

Strains of Pseudomonas fluorescens and Bacillus amyloliquefaciens, isolated from soybean rhizosphere, inhibited mycelial growth of Botrytis cinerea and Sclerotinia sclerotiorum, in vitro. Leaves from Brassica napus seedlings, pre-inoculated with either of these bacteria, exhibited systemic protection against fungal pathogens.     

Effect of Pseudomonas fluorescens RB4 and Bacillus subtilis 189 on the phytoremediation potential of Catharanthus roseus (L.) in Cu and Pb-contaminated soils

The remediation of heavy metal-contaminated soils has become a critical issue due to toxic effects of these metals on living organisms. The current research was conducted to study the effect of Pseudomonas fluorescens RB4 and Bacillus subtilis 189 on the growth and phytoremediation potential of Catharanthus roseus in Cu- and Pb-contaminated soils. The bacterial strains exhibited significantly higher level of water-extractable Pb and Cu in Pb, Cu, and Cu+Pb-contaminated. The P. fluorescens RB4 inoculated plants, produced 102%, 48%, and 45% higher fresh weight (FW) in soils contaminated with Cu, Pb, and both elements, respectively, as compared to un-inoculated control plants. Similarly, B. subtilis 189 inoculated plants produced 108%, 43%, and 114% more FW in the presence of Cu, Pb, and both elements. The plants co-cultivated with both bacteria exhibited 121%, 102%, and 177% higher FW, in Cu, Pb, and both elements contaminated soils, as compared to respective un-inoculated control. Co-cultivation of P. fluorescens RB4, B. subtilis 189, and P. fluorescens RB4 + B. subtilis 189 resulted in higher accumulation of Cu and Pb in shoots of the C. roseus grown in contaminated soils as compared to un-inoculated control. Bacterial treatments also improved the translocation and metal bioconcentration factors. The growth and phytoextraction capability of C. roseus was improved by inoculation of P. fluorescens RB4 and B. subtilis 189.     

Root bacteria from nematicidal plants and their biocontrol potential against trichodorid nematodes in potato

Trichodorid nematodes (Nematoda: Trichodoridae) are vectors of tobacco rattle virus (TRV), one of the causal agents of spraing disease in potato. Root bacteria from nematicidal plants and their control potential against Trichodoridae were the focus of this study. Bacteria isolated from the roots of 12 nematicidal plants and potato were characterized for their production of hydrolytic enzymes, hydrogen cyanide, phenol oxidation ability and antifungal activity towards the potato pathogen Rhizoctonia solani. Based on these functional traits, bacteria isolates were selected and tested in greenhouse conditions on potato (cv. Saturna) for their effect on plant growth, and screened for nematicidal activity against Paratrichodorus pachydermus and Trichodorus primitivus in naturally infested soil. Sixteen bacteria isolates out of 44 reduced nematode densities by 50–100%. Nine selected isolated were further tested by bacterizing potato tubers (cv. King Edward) which were planted in a trichodorid and TRV-infested soil. Four bacterial isolates consistently reduced nematode densities (by 56.7–74.4%) with no visual negative effect on plant growth. These isolates were tentatively identified, partly by fatty acid methyl ester (FAME) analysis as: Stenotrophomonas maltophilia, Bacillus mycoides, Pseudomonas sp., and one unidentified bacterium. The isolates originated from potato, Plantago major, Thymus vulgaris and Asparagus officinalis, respectively. Two Pseudomonas isolates obtained from Zinnia elegans and selected for their strong nematicidal activity in soil screening tests, did not reduce the nematode population when tested on potato. It is concluded that plants releasing nematicidal compounds may harbour nematode-antagonistic bacteria as well.     

Biosynthesis of Amino Acids from Hydrocarbons

Nine strains of bacteria, capable of synthesising amino acids from hydrocarbons, have been identified. Pseudomonas arvilla, Ps. fluorescens and Micrococcus ureae, previously reported as hydrocarbons assimilators, were found capable of synthesising amino acids. Pseudomonas taetrolens, Bacillus cereus var. mycoides, Bacillus subtilis and Bacillus sphaericus also synthesised amino acids from hydrocarbons.     

The effect of spent mushroom compost on Lecanicillium fungicola in vivo and in vitro

Leached spent mushroom compost (SMC) and its extract were tested to suppress Lecanicillium fungicola in white button mushroom. Sterile and non-sterile mixture of SMC and peat were used to assess suppressiveness against L. fungicola in greenhouse experiments. The extract of SMC was prepared with sterile, non-sterile, filtered, supplied with nystatin, streptomycin and penicillin antibiotics to evaluate their effect in suppression of pathogen in vitro. Isolated bacteria from SMC extract were tested for antagonism rate against Lecanicillium fungicola. The results of the experiments showed that all applications rate of none-sterile SMC were effective in control of pathogen. However, the sterile SMC amendments did not have a positive effect on the pathogen suppression in vitro or in vivo, as was expected. The treatments amended with SMC 100% and 60% showed the most suppressive effect in the control of pathogen. Using of non-sterile SMC 20%, 40%, 60% and peat soil were most effective in mushroom yield. The extract of leached SMC showed inhibition of L. fungicola in petri dishes. Three bacteria isolated from extract, Bacillus subtilis, Bacillus licheniformis and Bacillus amyloliquefacien identified using 16s rRNA, showed an antagonistic effect with the fungal growth.     

Numerical taxonomy of fluorescent Pseudomonas associated with tomato roots

The phenetic taxonomy of 110 fluorescent bacterial strains, isolated from the roots of tomatoes and other plants was numerically studied through 97 features including 69 assimilation tests. Thirty-two reference strains of various Pseudomonas spp. were additionally included. The strains clustered into 16 clusters at the 74% similarity level when using Jaccard similarity coefficients. Almost all field strains belonged to the P. fluorescens/P. putida-complex while none clustered with P. syringae and allied bacteria. The biovar II branch, as well as the newly described biovar VI of P. fluorescens, made up 55% and 20% respectively, of the field strains; two % were allocated to P. fluorescens biovar I and three % to biovar IV. Eleven % of the root associated strains were designated P. putida; six strains were biovar A, three strains biovar B while four strains could not be referred to any known biovar. The continuum within the P. fluorescens/P. putida-complex as well as the taxonomic status of the six biovars of P. fluorescens and the three biovars of P. putida are discussed.     

Effects of four Bacillus spp. of soil origin on the Colorado potato beetle Leptinotarsa decemlineata (Say)

Four species of Bacillus were isolated from soil in an effort to find safe, effective and alternative biological control agents against plant pests. These bacteria were identified as Bacillus pumilus, Bacillus sphaericus, Bacillus megaterium and Bacillus cereus on the basis of fatty acid methyl ester analysis and carbon utilization profiles by using Microbial Identification and Biolog Microplate Systems. Laboratory experiments carried out to determine the insecticidal activities of these isolates showed that B. pumilus caused 95.7 and 26.7% mortality and B. sphaericus caused 74.5 and 23.3% mortality of Leptinotarsa decemlineata larvae and adults, respectively. B. cereus and B. megaterium showed 51.1 and 29.7%, respectively, of L. decemlineata larvae. This study presents at least two Turkish isolates from the genus Bacillus showing high insecticidal activity against L. decemlineata.     

马铃薯疮痂病菌Streptomyces scabies拮抗细菌的筛选及BKS104鉴定

[背景] 近年来,马铃薯疮痂病的危害态势逐渐上升,在全国各主要产区均有发生,目前由于缺乏有效的防治手段,已造成了严重的经济损失。生物防治是防治土传病害的有效途径,逐渐成为了研究热点。[目的] 筛选对马铃薯疮痂病菌具有较高拮抗效果的菌株,为生防菌剂的开发提供菌种资源,同时也为马铃薯疮痂病的防治奠定理论基础。[方法] 采用平板对峙法和牛津杯试验法对分离得到的菌株进行初筛和复筛,并通过形态特征、生理生化特征及16S rRNA基因、gyrB基因序列分析结果对菌株进行鉴定。通过平板对峙法测定菌株的抑菌谱。[结果] 获得一株具有明显拮抗效果的菌株BKS104,抑菌直径达到43 mm,防效达到85%。其菌落圆形、乳白色、不透明、表面有光泽,边缘整齐,菌体杆状,革兰氏阳性菌。结合16S rRNA基因、gyrB基因的测序结果将其鉴定为贝莱斯芽孢杆菌,并对8种植物病原真菌均具有抑制效果。[结论] 菌株BKS104为贝莱斯芽孢杆菌,可以有效抑制马铃薯疮痂病菌的生长,安全性高,具有良好的生防潜力。     

Biological control of mushroom brown blotch disease using antagonistic bacteria

Button mushroom brown blotch disease is one of the most important and devastating diseases in Iran which is caused by Pseudomonas tolaasii. To screen antagonistic bacteria against this pathogen, major mushroom cultivation centres in Iran were surveyed and samples were taken from compost, soil cover and button caps with or without visible symptoms. In total, 120 bacteria were isolated on the basis of their morphology and pathology on excised tissue blocks of the fresh Agaricus bisporus. Among all tested bacteria, thirty-six isolates produced variable inhibition zones and reduced the symptom incidence on tissue blocks of A. bisporus. The most effective antagonistic bacteria caused almost 100% inhibition of pathogenic bacterium. To confirm the identification of antagonistic bacteria, RNA polymerase beta-subunit gene (rpoB) of five antagonistic strains (A1, A2, A3, A4 and A6) were amplified using primer sets of long amplicon primers (LAPS) and LAP27. The polymerase chain reaction products of the strains A1, A3 and A6 were sequenced. Based on phenotypic, biochemical and molecular characteristics, the bacterial antagonists were identified as P. putida (A1), P. reactants (A2 and A6), P. fluorescens (A3 and A4) and Bacillus subtilis (A5), respectively. In all four criteria including weight of the diseased and healthy caps, per cent disease severity and per cent disease incidence, the treatment 2 (P. fluorescens A4) was the most effective from among T1, T3, T4 and T5 treatments. Overall results of this study suggest that bacterial antagonists may be potential biocontrol agents for biological promotion of the health and growth of button mushroom.     

Bacterial inhibition of Orobanche aegyptiaca and Orobanche cernua radical elongation

The ability of different bacteria to inhibit Orobanche radical elongation was examined. Seeds of Orobanche aegyptiaca Pers. and O. cernua Loefl. were induced to germinate with the Growth Regulator GR24 in the presence or absence of test bacterium. Radical lengths were estimated microscopically on a scale relative to seed length (0–5 seed lengths) after 5 days of incubation at 25°C. The results indicated that Pseudomonas aeruginosa QUBC1, P. fluorescens QUBC3, Bacillus atrophaeus QUBC16, and B. subtilis QUBC18 significantly inhibited radical elongation (P≤0.01) of both O. aegyptiaca and O. cernua relative to control radicals, whereas Microbacterium hydrocarbonoxydans QUBC11 and Ochrobactrum anthropi QUBC13 showed less inhibitory effects. Other bacterial isolates had no inhibitory effects. Bacterial isolates were identified using the universal method in addition to morphological and biochemical features. The establishment of the inhibitory effect of the most promising isolates, B. atrophaeus QUBC16 and P. aeruginosa QUBC1 on radical elongation of both Orobanche spp. is a step towards utilizing such bacteria as biocontrol agents against O. aegyptiaca, O. cernua, and potentially other Orobanche species.     

Microbial manganese reduction mediated by bacterial strains isolated from aquifer sediments

One hundred and five strains isolated from aquifer sediments andEscherichia coli ML30S were tested for their ability to reduce manganese oxides. Eighty-two strains, includingE. coli, reduced manganese. In most cases the bacterial activity decreased the pH and Eh below 6.75 and 350 mV, respectively, enhancing a spontaneous and nonspecific reduction of manganese. However, for 12 strains the reduction was specifically catalyzed by bacteria; the high pH and Eh values would not permit a spontaneous reduction of manganese. Some of the most active strains were identified as genera common in soils and waters, i.e.,Pseudomonas, Bacillus, Corynebacterium, andAcinetobacter. Two strains were studied in detail. One of the strains, identified asPseudomonas fluorescens, required contact between the cells and the manganese oxides for reduction to occur. The reduction was inhibited by 15 mM of sodium azide. The other strain, identified asAcinetobacter johnsonii, catalyzed manganese reduction by an inductive and dialyzable substance which was excreted by the bacteria. The mechanism involved has not been previously demonstrated.     

Quorum quenching by Bacillus cereus U92: a double-edged sword in biological control of plant diseases

In the present survey, quorum quenching activity was examined from a biocontrol point of view. Acyl-homoserine lactone (AHL) degrading bacteria were isolated from tomato rhizosphere using two standard bioreporter strains and different synthetic AHLs and then identified according to 16S rDNA sequences. Five isolates capable of inactivating both short and long 3oxo-substituted AHLs showed high similarity with the genera Bacillus, Microbacterium and Arthrobacter, and thereby Bacillus cereus U92 was determined as the most efficient quorum quencher strain. In the quantitative experiments, this strain remarkably inactivated all synthetic AHLs up to 80%. In the laboratory co-cultures, B. cereus U92 efficiently quenched QS-regulated phenotypes in Agrobacterium tumefaciens, Pseudomonas aeruginosa, Pseudomonas chlororaphis and Chromobacterium violaceum. The strain successfully reduced the frequency of Ti-plasmid conjugal transfer in A. tumefaciens by about 99% in the binary cultures. Meanwhile, in a more natural environment, this strain acted as a biocontrol agent, efficient in alleviating QS-regulated crown gall incidence on tomato roots (up to 90%) as well as attenuating Pectobacterium soft rot on potato tubers (up to 60%). On the other hand, reducing phenazine production in P. chlororaphis operated as a suppressor of its QS-regulated biocontrol activity and also inhibited pyocyanin production in P. aeruginosa, a plant growth-promoting bacterium, by 75%. In general, B. cereus U92 seems very promising in the biological control of pathogenic bacteria; however, its broad AHL-degrading activity has a detrimental role on beneficial microbes which should not be neglected.