Using combinations of biocontrol agents to control Botrytis cinerea on strawberry leaves under fluctuating temperatures

Experiments were conducted with Botrytis cinerea on strawberry leaves to investigate where combinations of commercially available biological control agents (BCAs) might control B. cinerea more effectively than individual BCAs. Specifically, we studied the persistence of biocontrol activities, spread of BCAs among leaves, and biocontrol efficacy in relation to application regimes: mixed versus single BCA, pre-versus post-inoculation application, and sequential versus simultaneous application. Three BCA products (Sentinel, Serenade and Trianum) were used for this study. Overall, Serenade did not significantly reduce sporulation of B. cinerea on strawberry leaf discs whereas Sentinel and Trianum gave a similar and significant biocontrol efficacy. Biocontrol efficacy remained almost unchanged 10 days after application at 20/20°C (day/night) or 24/16°C temperature regimes. In contrast, reduced biocontrol efficacy at 26/14°C suggests BCA survival was reduced under these conditions. Incidence of B. cinerea sporulation on leaf discs was ca. 60% higher on leaves that emerged after the BCA application than on leaves directly exposed to BCA, indicating insufficient amount of the BCA had managed to spread to new leaves. Combinations of BCAs, whether applied simultaneously or sequentially (48 h apart), did not improve disease control over the most effective BCA within the combination applied alone. This indicated possible antagonism or interference between the BCAs. Results suggested that there was significant antagonism for most combinations of the three BCAs tested and the degree of antagonism increased as the time from BCA application to pathogen introduction lengthened.     

The development of ideas in biological control applied to forest products

After giving an overview of biological control agents and their application to forest products, the review considers the main biocontrol mechanisms that are thought to operate in wood. Competition for nutrients, antibiosis, mycoparasitism and production of extracellular enzymes have all been implicated in the biological control of wood decay fungi, although the relative importance of these potential mechanisms in vivo remains obscure. Attempts at biologically controlling basidiomycetes in forest products on a commercial scale have met with limited success and the environmental factors influencing efficacy are reviewed. It is concluded that the chances of achieving successful biocontrol of decay rely as much on appreciating the limitations of biological treatments as upon a comprehensive understanding of the mechanisms involved.     

Evaluation of biocontrol agents and potassium silicate for the management of powdery mildew of zucchini

Investigations were conducted under greenhouse and field conditions to evaluate the effects of potential biocontrol agents (BCAs) and soluble silicon (Si) on powdery mildew of zucchini caused by Podosphaera xanthii. Five BCAs were applied as foliar sprays to zucchini leaves and Si was drenched weekly into the rhizosphere of these plants.In the greenhouse, all BCAs provided significant control of powdery mildew with fungal isolates, reducing disease levels by up to 90%. Si alone reduced powdery mildew by as much as 35% and improved the efficacy of most of the biocontrol agents. Higher disease pressure reduced the efficacy of Si on powdery mildew but did not affect the performance of the BCAs. In the field, a disease reduction of 10–70% was achieved by BCAs and Si. Lower temperatures and high humidity ranges were suitable for optimal performances. The efficacy of the bacterial BCA, Serratia marcescens – B15 and silicon diminished at temperatures above 25 °C. The fungal BCAs (Clonostachys rosea – EH and Trichothecium roseum – H20) were better suited to higher temperatures (25–30 °C) and were tolerant of low RH values. Application of K₂SiO₂ to zucchini roots increased the level of Si in the leaves, which was responsible for suppression of the disease.     

Hrp?mutant bacteria as biocontrol agents: Toward a sustainable approach in the fight against plant pathogenic bacteria

Sustainable agriculture necessitates development of environmentally safe methods to protect plants against pathogens. Among these methods, application of biocontrol agents has been efficiently used to minimize disease development. Here we review current understanding of mechanisms involved in biocontrol of the main Gram-phytopathogenic bacteria-induced diseases by plant inoculation with strains mutated in hrp (hypersensitive response and pathogenicity) genes. These mutants are able to penetrate plant tissues and to stimulate basal resistance of plants. Novel protection mechanisms involving the phytohormone abscisic acid appear to play key roles in the biocontrol of wilt disease induced by Ralstonia solanacearum in Arabidopsis thaliana. Fully understanding these mechanisms and extending the studies to other pathosystems are still required to evaluate their importance in disease protection.     

Comparison of ATPase-Encoding Type III Secretion System hrcN Genes in Biocontrol Fluorescent Pseudomonads and in Phytopathogenic Proteobacteria

Type III protein secretion systems play a key role in the virulence of many pathogenic proteobacteria, but they also occur in nonpathogenic, plant-associated bacteria. Certain type III protein secretion genes (e.g., hrcC) have been found in Pseudomonas sp. strain SBW25 (and other biocontrol pseudomonads), but other type III protein secretion genes, such as the ATPase-encoding gene hrcN, have not been found. Using both colony hybridization and a PCR approach, we show here that hrcN is nevertheless present in many biocontrol fluorescent pseudomonads. The phylogeny of biocontrol Pseudomonas strains based on partial hrcN sequences was largely congruent with the phylogenies derived from analyses of rrs (encoding 16S rRNA) and, to a lesser extent, biocontrol genes, such as phlD (for 2,4-diacetylphloroglucinol production) and hcnBC (for HCN production). Most biocontrol pseudomonads clustered separately from phytopathogenic proteobacteria, including pathogenic pseudomonads, in the hrcN tree. The exception was strain KD, which clustered with phytopathogenic pseudomonads, such as Pseudomonas syringae, suggesting that hrcN was acquired from the latter species. Indeed, strain KD (unlike strain SBW25) displayed the same organization of the hrpJ operon, which contains hrcN, as P. syringae. These results indicate that the occurrence of hrcN in most biocontrol pseudomonads is not the result of recent horizontal gene transfer from phytopathogenic bacteria, although such transfer might have occurred for a minority of biocontrol strains.     

Biochanin-A ameliorates pulmonary fibrosis by suppressing the TGF-β mediated EMT,myofibroblasts differentiation and collagen deposition in in vitro and in vivo systems

Background: Idiopathic Pulmonary Fibrosis (IPF) is a progressive inflammatory disorder driven by a fibrotic cascade of events such as epithelial to mesenchymal transition, extracellular matrix production and collagen formation in the lungs in a sequential manner. IPF incidences were raising rapidly across the world. FDA approved pirfenidone and nintedanib (tyrosine kinase inhibitors) are being used as a first-line treatment drugs for IPF, however, neither the quality of life nor survival rates have been improved because of patient noncompliance due to multiple side effects. Thus, the development of novel therapeutic approaches targeting TGF-β mediated cascade of fibrotic events is urgently needed to improve the survival of the patients suffering from devastating disease.Purpose: The aim of this study was to investigate and validate the anti-fibrotic properties of Biochanin-A (isoflavone) against TGF-β mediated fibrosis in in vitro, ex vivo, in vivo models and to determine the molecular mechanisms that mediate these anti-fibrotic effects.Methods: The therapeutic activity of BCA was determined in in vitro/ex vivo models. Cells were pre-treated with BCA and incubated in presence or absence of recombinant-TGF-β to stimulate the fibrotic cascade of events. Pulmonary fibrosis was developed by intratracheal administration of bleomycin in rats. BCA treatment was given for 14 days from post bleomycin instillation and then various investigations (collagen content, fibrosis gene/protein expression and histopathological changes) were performed to assess the anti-fibrotic activity of BCA.Results: In vitro/ex vivo (Primary normal, IPF cell line and primary IPF cells/ Precision cut mouse lung slices) experiments revealed that, BCA treatment significantly (p < 0.001) reduced the expression of TGF-β modulated fibrotic genes/protein expressions (including their functions) which are involved in the cascade of fibrotic events. BCA treatment significantly (p < 0.01) reduced the bleomycin-induced inflammatory cell-infiltration, inflammatory markers expression, collagen deposition and expression of fibrotic markers in lung tissues equivalent or better than pirfenidone treatment. In addition, BCA treatment significantly (p < 0.001) attenuated the TGF-β1/BLM-mediated increase of TGF-β/Smad2/3 phosphorylation and resulted in the reduction of pathological abnormalities in lung tissues determined by histopathology observations.Conclusion: Collectively, BCA treatment demonstrated the remarkable therapeutic effects on TGF-β/BLM mediated pulmonary fibrosis using IPF cells and rodent models. This current study may offer a novel treatment approach to halt and may be even rescue the devastating lung scarring of IPF.     

Streptomyces sp. S160: a potential antagonist against chickpea charcoal root rot caused by Macrophomina phaseolina (Tassi) Goid

Biological control of charcoal root rot disease caused by Macrophomina phaseolina in chickpea was studied by using Streptomyces sp. S160. This biocontrol agent (BCA) inhibited the mycelial growth of M. phaseolina by 50 % in vitro and significantly reduced charcoal rot incidence in the greenhouse by 33.3 %. The greenhouse experiment revealed that seed treatment along with soil application supported the highest germination (88.6 %), vigor index (7326.91) and reduced root rot incidence (12.5 %) in comparison to seed treatment and soil application alone. BCA enhanced the growth and helped in inducing resistance against charcoal rot disease of chickpea caused by M. phaseolina by increasing activity of defense-related enzymes in chickpea plants, leading to the synthesis of defense chemicals in plants. BCA (Streptomyces sp. S160) was also characterized and identified by using polyphasic approaches including 16S rDNA sequencing.     

Evaluation of several commercial biocontrol products on European and North American populations of Phytophthora ramorum

Five commercially available biological control products were tested in vitro with seven isolates of Phytophthora ramorum from North American (NA1, NA2), and European (EU1) populations. The in vitro tests included dual culture methods and detached leaf assays on wounded Rhododendron and Camellia leaves. Variability in response to biocontrol agents among isolates of P. ramorum from North American and European populations was examined. In dual culture tests, both Bacillus subtilis products (Companion® and Serenade®) resulted in better inhibition of the NA1 group than NA2 and EU1. Actinovate® (Streptomyces lydicus) was the least effective of the three bacterial biocontrol agents and there was no difference in percent inhibition among P. ramorum lineages. Two products containing Trichoderma spp. were tested: Plant Helper® (T. atroviride) caused 100% inhibition of all lineages of P. ramorum, while SoilGard? (T. virens) was only about 30% effective. There was great variability among P. ramorum isolates in their response to biocontrol agents. All treatments reduced P. ramorum lesion size on both Rhododendron and Camellia. Combined treatments of Actinovate® with one other BCA did not perform as well as either treatment used individually. Best results were obtained with Serenade® on Rhododendron and Camellia foliage, especially against the NA1 group. Lack of a linear relationship between percent inhibition of P. ramorum by BCAs in vitro and foliar treatments on detached Rhododendron and Camellia leaves indicates that in vitro testing is a poor predictor of BCA performance on plant material.     

Attachment microbes antagonistic against Botrytis cinerea—biological control and scanning electron microscope studies in vivo

Bacteria and yeasts, selected by an attachment assay for their ability to adhere to Botrytis cinerea hyphae or conidia, were evaluated for biocontrol potential against B. cinerea on excised tomato stems. Eight of the 12 bacteria and seven of the eight yeast isolates conferred 90% to 100% biocontrol activity when antagonist populations were applied at three to 80 times the pathogen inoculum density. Biocontrol was maintained at similar levels when biocontrol agent (BCA) application was delayed up to 48 h after pathogen challenge. Scanning electron microscopy showed extensive colonisation of B. cinerea mycelium or conidia by many of these isolates and also evidence of pathogen degradation. The biocontrol efficacy and potential for the assay are discussed with respect to cell-to-cell adhesion as a vehicle to deliver antagonistic mechanisms to highly specific pathogen sites.     

Predicted ecological niches and environmental resilience of different formulations of the biocontrol yeast <Emphasis Type="Italic">Candida sake</Emphasis> CPA-1 using the Bioscreen C

Environmental resilience of biocontrol microorganisms has been a major bottleneck in the development of effective formulations. Candida sake is an effective biocontrol agent (BCA) against Penicillium expansum, Botrytis cinerea or Rhizopus stolonifer, and different formulations of the BCA have been optimised recently. The objective of this study was to compare the relative tolerance of different dry and liquid formulations of the biocontrol yeast C. sake CPA-1 to interacting environmental conditions using the Bioscreen C. Initially, the use of this automated turbidimetric method was optimised for use with different formulations of the biocontrol yeast. The best growth curves were obtained for the C. sake CPA-1 strain when grown in a synthetic grape juice medium under continuous shaking and with an initial concentration of 10⁵ CFUs ml^?1. All the formulations showed a direct relationship between optical density values and yeast concentrations. Temperature (15–30 °C) and water activity (a_w; 0.94–0.99) influenced the yeast resilience most profoundly, whereas the effect of pH (3–7) was minimal. In general, the liquid formulation grew faster in more interacting environmental conditions but only the yeast cells in the dry potato starch formulation could grow in some stress conditions. This rapid screening method can be used for effective identification of the resilience of different biocontrol formulations under interacting ecological abiotic conditions.     

The Saharan isolate Saccharothrix algeriensis NRRL B-24137 induces systemic resistance in Arabidopsis thaliana seedlings against Botrytis cinerea

Background and aim

Saccharothrix algeriensis NRRL B-24137, isolated from a Saharan soil, has been described as a potential biocontrol agent against Botrytis cinerea and other phytopathogens. However, the plant protection mechanisms involved still need to be described. The aim of this study was to determine this protection phenomenon as well as parts of the mechanisms involved, using Arabidopsis thaliana seedlings and B. cinerea.

Methods

The bacterial colonization process was evaluated on A. thaliana seedlings using fluorescence in situ hybridization. Protection of A. thaliana seedlings inoculated with NRRL B-24137 against B. cinerea was then evaluated. Parts of the mechanisms involved in the systemic protection against B. cinerea were evaluated using known mutants of genes involved in jasmonate (JA)/ethylene (ET)/salicylic acid (SA) signaling. Other Arabidopsis mutants, AtrhbohD-3, AtrhbohF-3, and ups1-1 were also screened to determine other parts of the mechanisms involved.

Results

The results showed that the strain NRRL B-24137 colonized, epi- and endophytically, the roots of Arabidopsis seedlings but the strain was not a systemic colonizer during the time of the experiment. The strain NRRL B-24137 also reduced B. cinerea symptoms and the protection was linked to known mechanisms of induced systemic resistance (ISR; JA/ET signaling), as well as to functionality of AtrbohF oxidase and of UPS1. Crosstalk between ET/JA and SA signaling could also be involved.

Conclusions

The isolate NRRL B-24137, after colonizing the root systems of A. thaliana, induces an ISR against B. cinerea, which is JA/ET dependent, but could also require SA crosstalk and protection could also require NAPDH oxidases and UPS1 functionalities.     

<Emphasis Type="Italic">Apis mellifera</Emphasis> and <Emphasis Type="Italic">Osmia cornuta</Emphasis> as carriers for the secondary spread of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> on apple flowers

The efficiency of two pollinators, Apis mellifera L. (Hymenoptera: Apidae) and the mason bee Osmia cornuta (Latreille) (Hymenoptera: Megachilidae), as carriers of biocontrol agents (BCA) from flower to flower (secondary colonisation) was investigated on apple cv ‘Golden Delicious’. The BCA tested was Bacillus subtilis, strain BD170 (Biopro®) developed for the control of the ‘fire blight’ caused by Erwinia amylovora (Burril) Winslow et al. The two insect species were studied as secondary BCA carriers on apple plants in pots under net screened tunnels. Their behaviour and capacity to deposit the BCA in the most receptive flower parts were compared both by washing, diluting and plating the flower organs on a recovery medium and by means of PCR analyses based on a molecular marker. O. cornuta showed better performances with respect to A. mellifera. For the field trials, pollinators were introduced in four apple orchards. During apple’s flowering, the BD170 (100 g hl^?l) was sprayed once in two fields, and twice in the others. The pollinators’ efficacy in carrying the BCA from sprayed flowers to the stigmas of newly opened ones at different times after the spray treatment was evaluated. The detection of the BCA was performed by PCR analysis. The percentages of positive PCR flower samples were higher in the internal treated areas of the fields with respect to the external untreated ones, but the high colonisation level found in the latter and in the flowers opened in both areas several days after the treatment(s) demonstrated that pollinators can play an important role as secondary carriers.     

Optimizing a Bacillus subtilis isolate for biological control of sugar beet cercospora leaf spot

Bacillus spp. have been used to control a number of leaf spot and post harvest diseases. Their capacity to form endospores facilitates long-term storage and relatively easy commercialization. This study focuses on optimizing a Bacillus subtilis isolate, BacB, for the control of sugar beet Cercospora leaf spot, caused by Cercospora beticola Sacc., by examining application timing, biocontrol agent (BCA) concentration, use of the selective nutrient substrate β-glucan, and the form of the BCA at time of application. A method for germinating endospores prior to spraying, without active aeration, is described. Examining the effects of varying β-glucan concentrations and levels of BacB at application demonstrated a complex interaction between β-glucan, BCA population, and disease control. In the 1998 field season, disease severity was significantly decreased, as compared to the control, at an application rate of 1×10⁶ CFU/ml, or higher, with 0% β-glucan. In 1999, there was less disease pressure, and all treatments reduced disease severity. Growth chamber experiments demonstrated that applying the bacteria as vegetative cells instead of spores or applying the BCA 1–5 days before infection could significantly increase disease control. Laboratory experiments demonstrated the ability to induce germination and vegetative growth of BacB from a spore formulation, without shaking or fermentation equipment. This shows promise for optimizing Bacillus sp. for biological control. In field trials the vegetative cells did not perform better than the spore application, though the potential for β-glucan to increase disease was demonstrated.     

Incorporation into the transplant soil plug of the plant protective agent Paenibacillus alvei strain K165 confers protection to melon against Fusarium wilt

Fusarium oxysporum f.sp. melonis (FOM) is a plant pathogen affecting melon production worldwide. An environmental friendly disease management strategy is the use of biocontrol agents (BCAs). Towards this direction, two BCA release strategies, seed coating and amendment of the transplant soil plug with the BCA strain Paenibacillus alvei K165 at various ratios, were evaluated against FOM in planta and in vitro. A reduction in Fusarium wilt symptom development was observed in melon plants, after mixing the transplant soil plug with K165 (10⁷ cfu g^?1 powder) at a ratio of 10 % (v/v). The monitoring of K165 rhizosphere population in the different treatments revealed a possible existence of a threshold population level that has to be attained before suppression of disease occurs. The data of the present study suggest that K165 plant protective activity against FOM can be possibly attributed to antibiosis and the triggering of Chit1 and Pal1 gene expression.     

BCA3 contributes to the malignant progression of hepatocellular carcinoma through AKT activation and NF-κB translocation

Hepatocellular carcinoma (HCC) is one of the most lethal malignancies worldwide with elusive molecular mechanisms. The aim of this study is to investigate the clinical significance and biological roles of breast cancer-associated protein 3 (BCA3) in HCC. Our investigation demonstrated that BCA3 expression was up-regulated in primary HCC tissues, and BCA3 levels were positively correlated with tumor size, TNM stage, microvascular invasion and poor prognosis. BCA3 promoted tumor growth, metastasis and angiogenesis of HCC in vitro and in vivo. Moreover, we found that BCA3 induced aggressive behaviors were mediated by AKT activation, which in turn activated mTOR signalling pathway and induced cytoplasm-nuclear translocation of NF-κB p65. Blockage of AKT signalling pathway by a specific AKT inhibitor LY294002 impaired BCA3 mediated phenotypes. Collectively, our current study indicated the pleiotropic effects of BCA3 in HCC progression, and blockage of BCA3-AKT pathway might contribute to development of therapeutic measures for HCC.     

Effects of actinobacteria on plant disease suppression and growth promotion

Biological control and plant growth promotion by plant beneficial microbes has been viewed as an alternative to the use of chemical pesticides and fertilizers. Bacteria and fungi that are naturally associated with plants and have a beneficial effect on plant growth by the alleviation of biotic and abiotic stresses were isolated and developed into biocontrol (BCA) and plant growth-promoting agents (PGPA). Actinobacteria are a group of important plant-associated spore-forming bacteria, which have been studied for their biocontrol, plant growth promotion, and interaction with plants. This review summarizes the effects of actinobacteria as BCA, PGPA, and its beneficial associations with plants.     

Cultivation-Independent Analysis of Fungal Genotypes in Soil by Using Simple Sequence Repeat Markers

Cultivation-independent analyses of fungi are used for community profiling as well as identification of specific strains in environmental samples. The objective of the present study was to adapt genotyping based on simple sequence repeat (SSR) marker detection for use in cultivation-independent monitoring of fungal species or strains in bulk soil DNA. As a model system, a fungal biocontrol agent (BCA) based on Beauveria brongniartii, for which six SSR markers have been developed, was used. Species specificity of SSR detection was verified with 15 fungal species. Real-time PCR was used to adjust for different detection sensitivities of the six SSR markers as well as for different template quantities. The limit for reliable detection per PCR assay was below 2 pg target DNA, corresponding to an estimated 45 genome copies of B. brongniartii. The cultivation-independent approach was compared to cultivation-dependent SSR analysis with soil samples from a B. brongniartii BCA-treated field plot. Results of the cultivation-independent method were consistent with cultivation-dependent genotyping and allowed for unambiguous identification and differentiation of the applied as well as indigenous strains in the samples. Due to the larger quantities of soil used for cultivation-dependent analysis, its sensitivity was higher, but cultivation-independent SSR genotyping was much faster. Therefore, cultivation-independent monitoring of B. brongniartii based on multiple SSR markers represents a rapid and strain-specific approach. This strategy may also be applicable to other fungal species or strains for which SSR markers have been developed.     

Herbivore-induced shifts in carbon and nitrogen allocation in red oak seedlings

* A dual-isotope, microcosm experiment was conducted with Quercus rubra (red oak) seedlings to test the hypothesis that foliar herbivory would increase belowground carbon allocation (BCA), carbon (C) rhizodeposition and nitrogen (N) uptake. Plant BCA links soil ecosystems to aboveground processes and can be affected by insect herbivores, though the extent of herbivore influences on BCA is not well understood in woody plants. * Microcosms containing 2-yr-old Q. rubra seedlings and soil collected from the Coweeta Hydrologic Laboratory (NC, USA) were subjected to herbivory or left as undamaged controls. All microcosms were then injected with 15N-glycine and pulsed with 13CO2. * Contrary to our hypothesis, herbivore damage reduced BCA to fine roots by 63% and correspondingly increased allocation of new C to foliage. However, 13C recoveries in soil pools were similar between treatments, suggesting that exudation of C from roots is an actively regulated component of BCA. Herbivore damage also reduced N allocation to fine roots by 39%, apparently in favor of storage in taproot and stem tissues. * Oak seedlings respond to moderate insect herbivore damage with a complex suite of allocation shifts that may simultaneously increase foliar C, maintain C rhizodeposition and N assimilation, and shift N resources to storage.     

Is Puccinia xanthii a suitable biological control agent of Ambrosia artemisiifolia?

Common ragweed, Ambrosia artemisiifolia, is a highly allergenic North American plant that has become invasive in some parts of Europe, Asia and Australia following its introduction to many places in the world. Some earlier works suggested that a microcyclic autoecious rust fungus, Puccinia xanthii, known to infect A. artemisiifolia in the USA only, can be considered as a potential classical biocontrol agent (BCA) of this noxious weed in Europe and elsewhere. However, an extensive field survey did not reveal the presence of either P. xanthii or any other rusts on common ragweed in 14 US states and two Canadian provinces in 2002 and 2003. Moreover, P. xanthii infecting A. artemisiifolia has never been recorded in Canada, although it is known to occur on A. trifida and Xanthium spp. there. Nevertheless, herbarium specimens collected between 1855 and 1963 in five states of the USA confirmed the presence of P. xanthii on A. artemisiifolia. It is concluded that currently P. xanthii cannot be regarded as a promising BCA of A. artemisiifolia, although it did occur on common ragweed at least a few decades ago in the USA and some forms of this rust species have already been evaluated as effective BCAs of Xanthium in Australia.