Use Of Biocontrol Agents Research Articles

Streptomyces roseolus, A Promising Biocontrol Agent Against Aspergillus flavus, the Main Aflatoxin B₁ Producer.

Crop contamination by aflatoxin B1 is a current problem in tropical and subtropical regions. In the future, this contamination risk may be expanded to European countries due to climate change. The development of alternative strategies to prevent mycotoxin contamination that further contribute to the substitution of phytopharmaceutical products are thus needed. For this, a promising method resides in the use of biocontrol agents. Several actinobacteria strains have demonstrated to effectively reduce the aflatoxin B1 concentration. Nevertheless, the molecular mechanism of action by which these biological agents reduce the mycotoxin concentration has not been determined. The aim of the present study was to test the potential use of Streptomyces roseolus as a biocontrol agent against aflatoxin B1 contamination. Co-cultures with Aspergillus flavus were conducted, and the molecular fungal response was investigated through analyzing the q-PCR expression of 65 genes encoding relevant fungal functions. Moreover, kojic and cyclopiazonic acid concentrations, as well as morphological fungal changes were also analyzed. The results demonstrated that reduced concentrations of aflatoxin B1 and kojic acid were respectively correlated with the down-regulation of the aflatoxin B1 gene cluster and kojR gene expression. Moreover, a fungal hypersporulated phenotype and a general over-expression of genes involved in fungal development were observed in the co-culture condition.

Evolution of the biocontrol active substances in the framework of the European Pesticide Regulation (EC) No. 1107/2009.

The use of biocontrol agents (BCA) is growing strongly with a market of €1.9billion worldwide and €542million in Europe, and an estimated annual growth of between 15% and 20%. Biocontrol substances use natural mechanisms as part of integrated pest management. With the exception of macro-organisms, substances or products comprising microorganisms, semiochemicals and natural substances depend on plant protection Regulation (EC) No. 1107/2009. When this regulation came into force, a number of BCA had already been approved. The total number of BCA rose during the period 2011-2018 to ∼60 substances in line with global growth in approved substances. This growth was not smooth or equal between biocontrol categories; the microorganisms category increased the most, with a doubling of the number of approved substances in use, followed by natural substances and semiochemicals. This positive development has been influenced by many factors such as scientific advances and environmental characteristics. However, some obstacles remain that limit the development of BCA (market size, variability in effectiveness, etc.). Regulatory measures could be a key to enhancing the development of biocontrol registrations. © 2018 Society of Chemical Industry.

Antifungal Activity of Bacillus Species Against Fusarium and Analysis of the Potential Mechanisms Used in Biocontrol.

Fusarium is a complex genus of ascomycete fungi that consists of plant pathogens of agricultural relevance. Controlling Fusarium infection in crops that leads to substantial yield losses is challenging. These economic losses along with environmental and human health concerns over the usage of chemicals in attaining disease control are shifting focus toward the use of biocontrol agents for effective control of phytopathogenic Fusarium spp. In the present study, an analysis of the plant-growth promoting (PGP) and biocontrol attributes of four bacilli (Bacillus simplex 30N-5, B. simplex 11, B. simplex 237, and B. subtilis 30VD-1) has been conducted. The production of cellulase, xylanase, pectinase, and chitinase in functional assays was studied, followed by in silico gene analysis of the PGP-related and biocontrol-associated genes. Of all the bacilli included in this study, B. subtilis 30VD-1 (30VD-1) demonstrated the most effective antagonism against Fusarium spp. under in vitro conditions. Additionally, 100 μg/ml of the crude 1-butanol extract of 30VD-1’s cell-free culture filtrate caused about 40% inhibition in radial growth of Fusarium spp. Pea seed bacterization with 30VD-1 led to considerable reduction in wilt severity in plants with about 35% increase in dry plant biomass over uninoculated plants growing in Fusarium-infested soil. Phase contrast microscopy demonstrated distortions and abnormal swellings in F. oxysporum hyphae on co-culturing with 30VD-1. The results suggest a multivariate mode of antagonism of 30VD-1 against phytopathogenic Fusarium spp., by producing chitinase, volatiles, and other antifungal molecules, the characterization of which is underway.

Expression of defense-related genes in mung bean varieties in response to Trichoderma virens alone and in the presence of Rhizoctonia solani infection.

Web blight/wet root rot caused by Rhizoctonia solani is one of the major constraints for mung bean (Vigna radiata) production. Growing of resistant varieties and use of biocontrol agents are the feasible options available to manage the disease. The present study was conducted to determine the variation in the expression of various defense-related genes in susceptible and resistant mung bean varieties in response to biocontrol agent Trichoderma virens and R. solani interactions. The primers were designed using sequences of defense-related genes, namely PR 10, epoxide hydrolase (EH), catalase and calmodulin available in NCBI database and evaluated against cDNA obtained from both susceptible and resistant mung bean plants at 1-4days post-inoculation (dpi) with the test pathogen R. solani and biocontrol agent T. virens using conventional PCR and qPCR analyses. R. solani inoculation upregulated the mean expression of PR 10 and calmodulin in susceptible and resistant varieties, respectively, whereas downregulated in the rest of the treatments. Quantitative PCR analysis showed that except catalase in the susceptible variety, which is downregulated, the expression of PR 10, EH, catalase and calmodulin was upregulated in both resistant and susceptible varieties in response to T. virens alone and in the presence of R. solani. In general, the expression of PR 10 and calmodulin was highest at 1dpi whereas EH and catalase expression were maximum at 4dpi. The application of T. virens suppressed the development of disease in the presence of R. solani in both susceptible and resistant varieties with more pronounced effect in resistant variety. Thus, the application of biocontrol agent T. virens upregulated the expression of defense-related genes and reduced disease development.

Characterization of culturable gut bacterial isolates from wild population of melon fruit fly (Bactrocera cucurbitae) and assessing their attractancy potential for sustainable pest management

The melon fruit fly, Bactrocera cucurbitae (Coquillett) poses a serious threat to cucurbit production worldwide. Its management by conventional means remains difficult due to their proclivity to oviposite in fruit. In view of the increasing environmental safety concerns, use of biocontrol agents for sustainable pest control holds immense potential. Given this, the study intended to identify the cultivable bacteria inhabiting the intestinal tract of adult male and female melon fruit flies (Bactrocera cucurbitae) separately from field-collected population, and to assess the attractiveness of these bacteria to the fly. All selected bacterial isolates were identified and characterized based on morphological, biochemical and 16S rRNA gene sequence. Bacterial community identified in the gut of B. cucurbitae predominantly composed of Enterobacteriaceae followed by Staphylococcaceae, Enterococcaceae, Bacillaceae and Brucellaceae. Further, the laboratory bioassay was employed to examine the attractiveness of the supernatant as well as whole culture broth of 10 different species of bacteria to B. cucurbitae adults. Among these, Klebsiella oxytoca and Citrobacter freundii followed by Bacillus cereus were found highly attractive to fruit flies. The field experiment using supernatant of two bacteria demonstrated that the K. oxytoca was significantly more attractive to female flies followed by C. freundii.

Jumbo Bacteriophages Are Represented Within an Increasing Diversity of Environmental Viruses Infecting the Emerging Phytopathogen, Dickeya solani.

Dickeya species are economically important phytopathogens widespread in mainland Europe that can reduce crop yields by 25%. There are no effective environmentally-acceptable chemical systems available for diseases caused by Dickeya. Bacteriophages have been suggested for use in biocontrol of these pathogens in the field, and limited field trials have been conducted. To date the majority of bacteriophages capable of infecting Dickeya solani, one of the more aggressive species, are from the same family, the Ackermannviridae, many representatives of which have been shown to be unsuitable for use in the field due to their capacity for generalized transduction. Members of this family are also only capable of forming individual plaques on D. solani. Here we describe novel bacteriophages from environmental sources isolated on D. solani, including members of two other viral families; Myoviridae and Podoviridae, most of which are capable of forming plaques on multiple Dickeya species. Full genomic sequencing revealed that the Myoviridae family members form two novel clusters of jumbo bacteriophages with genomes over 250 kbp, with one cluster containing phages of another phytopathogen Erwinia amylovora. Transduction experiments showed that the majority of the new environmental bacteriophages are also capable of facilitating efficient horizontal gene transfer, however the single Podoviridae family member is not. This particular phage therefore has potential for use as a biocontrol agent against multiple species of Dickeya.

Comparative effects of temperature and thermoregulation on candidate strains of entomopathogenic fungi for Moroccan locust Dociostaurus maroccanus control

Strains IMI 330189 of Metarhizium acridum (Driver & Milner) J.F. Bisch., Rehner & Humber (Hypocreales: Clavicipitaceae) and EABb 90/2-Dm of Beauveria bassiana (Bals.-Criv.) Vuill. (Hypocreales: Cordycipitaceae) are a promising biocontrol tool of Dociostaurus maroccanus (Thunberg) (Orthoptera: Acrididae), although the effects of thermoregulation and Moroccan locust-fever on the infection process of these fungi remain unknown. In vitro experiments, measuring conidial germination and hyphal growth either at constant or at fluctuating temperatures simulating thermoregulatory conditions, indicated that strain IMI 330189 had greater fitness at temperatures above 27 °C and the strain EABb 90/2-Dm was better adapted to the temperature range of 10–25 °C. These effects were mirrored in vivo, where locust thermoregulation caused a marked reduction in the virulence of EABb 90/2-Dm in comparison to no thermoregulation conditions (average survival time: 6.10 vs. 15.83 days; mortality: 100% vs. 73.7%) but only a moderate reduction in the virulence of IMI 330189 (average survival time: 4.57 vs. 8.26 days; mortality: 100% vs. 100%). Thermal gradient experiments revealed that the strain IMI 330189 induced behavioral fever in D. maroccanus (preferred temperatures approximately 4 °C above the uninfected control), although it only led to a slight reduction in virulence. Strain EABb 90/2-Dm did not induce such a clear behavioral response. Under the temperature conditions of the main breeding areas of the Moroccan locust, strain IMI 330189 is likely a better candidate for use in biocontrol, although strain EABb 90/2-Dm could be also a good alternative in more temperate environments either as a stand-alone one or in mixed combinations of the two fungal strains, potentially providing more effective control over a broader range of temperatures.

Systemic method to isolate large bacteriophages for use in biocontrol of a wide-range of pathogenic bacteria

Large phages are characterized by genomes around 200kbp or more. They can infect wide host ranges of bacteria and maintain long-lasting infection. There is no standard method for selective isolation of large phages. In this study, we developed a systemic method to isolate large phages and succeeded in isolating 11 large phages, named Escherichia phage E1∼E11. Electron microscopy observations revealed typical Myoviridae phages with big capsids and long contractile tails. Genome sizes of the isolated phages were determined by pulsed-field gel electrophoresis and found to be in two groups, those around 200kbp for E1, E2, E5, E6, E7, E9 and E10 phages, and others of approximately 450kbp for E3, E4, E8 and E11 phages. The isolated large phages had wide host ranges: for example, E9 was effective against Shigella sonnei SH05001, Shigella bydii SH00007, Shigella flexneri SH00006, Salmonella enterica serovar Enteritidis SAL01078 and Escherichia coli C3000 (K-12 derivative), as well as its original host E.coli BL21. Screening of these jumbo phages was performed with non-pathogenic E.coli strains as hosts. Therefore, this method opens a way to isolate jumbo phages infecting wide ranges of pathogenic bacteria in a typical laboratory with standard laboratory strains as the hosts. The isolated large phages will be good candidates for biocontrol of various pathogens.

Induction of resistance to Meloidogyne incognita by DL-Beta amino butyric acid under salt stress condition

Root-knot nematodes cause severe yield losses in crops. Though chemicals have been widely used to manage them, these chemicals have been restricted due to their hazardous environmental and health effects. In recent years, the use of bio-control agents and the induction of resistance are prominent control methods. In the present study, DL-β-Amino Butyric Acid (BABA) was tested to control Meloidogyne incognita under salt stress conditions. The results indicate that 100 mM NaCl in combination with BABA (125 μg/ml) significantly reduced gall index by 45% compared to the control. Interestingly, the lower dose of BABA combined with abiotic stress resulted in a similar efficacy to the higher dose of BABA (250 μg/ml).

Assessing synergism of combined applications of Trichoderma harzianum and Pseudomonas fluorescens to control blast and bacterial leaf blight of rice

Combined application of biocontrol agents (BCAs) have been used to exploit their synergistic potential. But the analysis of additive, synergistic and antagonistic effects of BCAs has rarely been explored for the biocontrol of plant diseases. This study was conducted to evaluate in vitro and in vivo biocontrol potential of the combined application of Trichoderma harzianum strain Th3 and Pseudomonas fluorescens strain RRb11, and the fungicide carbendazim against the rice blast (RB) pathogen, Magnaporthe oryzae, and the bacterial leaf blight (BLB) pathogen, Xanthomonas oryzae pv. oryzae. Combined application of T. harzianum Th3 and P. fluorescens RRb11 synergistically reduced severity of RB by 69.5% in comparison to the untreated control, displaying a synergy factor (SF) of 1.29. P. fluorescens RRb 11 alone and in combination with T. harzianum Th3 and the mixture of T. harzianum Th3 + carbendazim was antagonistic (SF = 0.71 and 0.45, respectively). The combined application of T. harzianum Th3 and P. fluorescens RRb 11 also enhanced several rice plant growth and yield parameters. Additive, synergistic or antagonistic effects among the two BCAs were assessed by applying the numerical hypothesis of ‘Bliss Independence’ which suggested that the combined application of the two BCAs was synergistic in reducing RB but antagonistic with respect to BLB. Out of the six combined application interactions for both the diseases only one interaction (T. harzianum Th3 and P. fluorescens RRb11) was synergistic against RB, which suggested that synergism is a rare event in combined use of BCAs.

Conidia and blastospores of Metarhizium spp. and Beauveria bassiana s.l.: Their development during the infection process and virulence against the tick Rhipicephalus microplus

The current study compared the virulence of conidia and blastospores of Metarhizium robertsii (IP 146), M. anisopliae sensu lato (s.l.) (IP 363) and Beauveria bassiana s.l. (IP 361 and CG 307) against unfed larvae and engorged females of Rhipicephalus microplus (Acari: Ixodidae). In addition, the development of fungal propagules on tick cuticle was investigated. Tick larvae were treated with fungal suspensions at 106, 107 or 108 propagules mL−1, and percent mortality was assessed every two days. Engorged females were immersed in fungal suspensions (1.0 × 107 propagules mL−1) for 1 min, and their biological parameters monitored daily. The virulence of conidia and blastospores against larvae varied considerably among the isolates tested. Only females treated with blastospores of IP 146 or IP 361 presented lower nutrient and egg production indices than the control group; the higher percent control was reached when females were treated with blastospores of IP 146 (98%), IP 363 (79%), or IP 361 (93%) in comparison to the groups treated with conidia, 71%, 59%, or 63%, respectively. Engorged females treated with conidia or blastospores of IP 146 or IP 361 were also examined by scanning electron microscopy (SEM). Germination of blastospores of IP 146 and IP 361 was observed on tick cuticle after 4 h incubation at 27 ± 1 °C and RH > 90%, whereas germ tubes from conidia of both isolates were observed at 48 h. Appressoria in developing blastospores of B. bassiana IP 361 were observed after 4 h incubation, whereas no appressoria were seen in developing blastospores of M. robertsii IP 146. Blastospore penetrations by both fungal isolates through natural openings was also evidenced by SEM; fine sections of R. microplus engorged females treated with blastospores of IP 146 or IP 361 confirmed that these isolates penetrated through their cuticle and natural openings. Blastospores might be promising for use in biocontrol of ticks, since they are virulent against R. microplus and present rapid development on their cuticle.

Genomic analysis and immune response in a murine mastitis model of vB_EcoM-UFV13, a potential biocontrol agent for use in dairy cows

Bovine mastitis remains the main cause of economic losses for dairy farmers. Mammary pathogenic Escherichia coli (MPEC) is related to an acute mastitis and its treatment is still based on the use of antibiotics. In the era of antimicrobial resistance (AMR), bacterial viruses (bacteriophages) present as an efficient treatment or prophylactic option. However, this makes it essential that its genetic structure, stability and interaction with the host immune system be thoroughly characterized. The present study analyzed a novel, broad host-range anti-mastitis agent, the T4virus vB_EcoM-UFV13 in genomic terms, and its activity against a MPEC strain in an experimental E. coli-induced mastitis mouse model. 4,975 Single Nucleotide Polymorphisms (SNPs) were assigned between vB_EcoM-UFV13 and E. coli phage T4 genomes with high impact on coding sequences (CDS) (37.60%) for virion proteins. Phylogenetic trees and genome analysis supported a recent infection mix between vB_EcoM-UFV13 and Shigella phage Shfl2. After a viral stability evaluation (e.g pH and temperature), intramammary administration (MOI 10) resulted in a 10-fold reduction in bacterial load. Furthermore, pro-inflammatory cytokines, such as IL-6 and TNF-α, were observed after viral treatment. This work brings the whole characterization and immune response to vB_EcoM-UFV13, a biocontrol candidate for bovine mastitis.

Phenotypic Variation and Genomics of Bdellovibrio Predatory Bacteria from the Built Environment

Bdellovibrio are predatory bacteria that attack and kill Gram‐negative bacteria. They are found in a range of environments, including soil, water, and living organisms, but little is known about Bdellovibrio in the built environment. Surfaces, such as drains, are ideal for biofilm formation, which may provide stable prey sources for predatory bacteria. Investigating Bdellovibrio from the built environment will broaden our understanding of variation in prey range and predation efficiency and contribute to the development of predatory bacteria as biocontrol agents for use in settings such as hospital buildings.To test for the presence of predatory bacteria in built environment sites, we collected swab samples, extracted metagenomic DNA, and performed PCR with Bdellovibrio‐specific primers. Using this approach, we detected evidence of Bdellovibrio in a janitorial closet drain at Providence College. To obtain pure isolates, we made enrichments by combining swab samples from the drain with either Raoultella, a Gram‐negative bacterium previously isolated from a freshwater site, or E. coli ML35. Six predatory bacteria isolates were obtained using Raoultella as prey, and one was obtained using E. coli. Plaque features of each isolate on double agar overlay plates varied in size, shape, and appearance.To classify the isolates, we sequenced the 16S rRNA gene and found that the ~1300 bp sequence was identical for all isolates. This 16S rRNA gene sequence has 95%‐96% identity to Bdellovibrio bacteriovorus, suggesting the isolates are within the Bdellovibrio genus. We then generated short read sequencing data for each isolate using Illumina and performed de novo assembly using SPAdes. These assemblies yielded 2–11 contigs. Using the isolate with two contigs as a reference, we aligned each draft assembly and found at least 99% nucleotide identity across the full length of each contig. Given the identical 16S rRNA genes and the high sequence similarity among draft genome assemblies, this suggests that all seven Bdellovibrio isolates from the drain are representatives of a clonal population, but they are demonstrating plasticity in plaque phenotype. An alternative hypothesis is that small differences in gene sequences or gene content underlie the variation in plaque phenotype. To test these hypotheses, we are working on generating a complete genome using long read sequencing with PacBio.To understand variation in prey range, we challenged three of the Bdellovibrio isolates from the drain with eight Gram‐negative prey. Each isolate attacked the same two prey strains (Raoultella and E. coli ML35). This narrow prey range suggests that these Bdellovibrio may be prey specialists. We are isolating multiple prey strains from the drain to provide insight into plaque phenotype and prey range variation. Moving forward we will test predation efficiency using a variety of Gram‐negative bacteria. This work will contribute to our understanding and development of predatory bacteria and may inform their use in biofilm control and as an antibiotic alternative.Support or Funding InformationResearch reported in this poster was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number 2 P20 GM103430 and startup funding from Providence College.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The mycoparasitic fungus Clonostachys rosea responds with both common and specific gene expression during interspecific interactions with fungal prey.

Clonostachys rosea is a necrotrophic mycoparasitic fungus, used for biological control of plant pathogenic fungi. A better understanding of the underlying mechanisms resulting in successful biocontrol is important for knowledge‐based improvements of the application and use of biocontrol in agricultural production systems. Transcriptomic analyses revealed that C. rosea responded with both common and specific gene expression during interactions with the fungal prey species Botrytis cinerea and Fusarium graminearum. Genes predicted to encode proteins involved in membrane transport, biosynthesis of secondary metabolites and carbohydrate‐active enzymes were induced during the mycoparasitic attack. Predicted major facilitator superfamily (MFS) transporters constituted 54% of the induced genes, and detailed phylogenetic and evolutionary analyses showed that a majority of these genes belonged to MFS gene families evolving under selection for increased paralog numbers, with predicted functions in drug resistance and transport of carbohydrates and small organic compounds. Sequence analysis of MFS transporters from family 2.A.1.3.65 identified rapidly evolving loop regions forming the entry to the transport tunnel, indicating changes in substrate specificity as a target for selection. Deletion of the MFS transporter gene mfs464 resulted in mutants with increased growth inhibitory activity against F. graminearum, providing evidence for a function in interspecific fungal interactions. In summary, we show that the mycoparasite C. rosea can distinguish between fungal prey species and modulate its transcriptomic responses accordingly. Gene expression data emphasize the importance of secondary metabolites in mycoparasitic interactions.

Temperature and Exposure Time in Cold Storage Reshape Parasitic Performance of Habrobracon hebetor (Hymenoptera: Braconidae).

Cold storage can extend shelf life of parasitoids for use in biocontrol. However, cold storage may have negative impacts on life history traits of the parasitoids and, therefore, on their performance as biocontrol agents. Here, we examine the effect of cold storage on life history traits of Habrobracon hebetor (Say) (Hymenoptera: Braconidae), a parasitoid of several economic lepidopteran pests. Newly emerged wasps were stored at three constant temperatures (3°C, 5°C, 7°C) for up to 4 wk. Both temperature and exposure time significantly affected longevity, parasitism, fecundity, and sex ratio. Significant reduction in longevity was observed at 3°C and 7°C, whereas longevity of wasps stored at 5°C remained stable up to the second week and then gradually decreased in Weeks 3 and 4. Parasitism rate also significantly decreased after cold storage at 3°C, 5°C, and 7°C (ranked from high to low). Fecundity decreased at T 3°C and T 5°C but this trait was not affected by storage at T 7°C. A significant shift in male production was observed at T 5°C in Week 3, but in Week 4, the only treatment with male biased reproduction was T 3°C. These results show that the effect of temperature and exposure time in cold storage is trait dependent. Overall, storage at 5°C for a period of 3 wk least impacted most life-history traits of H. hebetor wasps.

Impact of Rhizophagus irregularis MUCL 41833 on disease symptoms caused by Phytophthora infestans in potato grown under field conditions

In organic potato production in Europe, only copper-based fungicides allow to directly control Phytophthora infestans (the causal agent of late blight). Due to environmental concerns caused by the repeated and excessive use of Cu before the nineties, the EU legislation has promoted alternatives approaches such as the use of biocontrol agents (e.g. arbuscular mycorrhizal fungi – AMF). Here, two field trials were conducted over two climatic-contrasting growing seasons. Trial 1 was characterized by a dry and hot cultural season with low pressure of P. infestans, while trial 2 was conducted under high humidity and relatively low temperatures with high pressure of the pathogen. In both trials, sprouted potato tubers were inoculated with AMF in the greenhouse before transplanting to the field. A Real-Time quantitative PCR assay was designed to target the inoculant strain Rhizophagus irregularis MUCL 41833 as well as the native Rhizophagus irregularis strains. In both trials, the inoculated AMF was detected in the roots at harvest, demonstrating the capacity of the inoculated strain to incorporate the microbiome of the potato plants. In the first trial, disease severity in AMF pre-colonized potato plants was markedly decreased and the onset of late blight symptoms was delayed by 10 days. In contrast, in the second trial no differences were noticed between AMF pre-colonized and control plants. In both trials, no mycorrhizal effect was noticed on tuber yield. As a conclusion, disease severity of P. infestans, measured by symptoms development on leaves, was decreased in AMF pre-colonized plants under conditions of low pressure of late blight and over a short period of time, while under conditions more adequate to the pathogen, no reduction in symptoms was noticed.

Streptomyces spp. as biocontrol agents against Fusarium species.

Abstract Streptomycetes are the largest taxon of antibiotic producers in the microbial world. Nevertheless, they are less studied than other biocontrol agents against Fusarium diseases and, perhaps, plant diseases in general. Plant diseases incited by Fusarium species are notably challenging. Four species complexes in the genus are pathogenic: Fusarium fujikuroi , F. graminearum , F. solani and F. oxysporum . Being a vascular pathogen, F. oxysporum is particularly difficult to control by using microbial antagonists. Most research has remained at early experimental stages, and only few Streptomyces spp. strains have been assayed under diverse conditions. Few commercial products consisting of Streptomyces spp. have often provided fluctuating results across trials. Five biocontrol trials conducted in the field using streptomycetes report reductions of diverse Fusarium wilts by 0-55%, with one case (cucumber) of yield increase by 1.3-fold. Among 38 articles dealing with pot-experiments, 16 report a disease control level above 50%, seven of which with a level above 70%. The chitinolytic activity of Streptomyces spp. strains plays an important role in the biocontrol of Fusarium diseases, and the plant growth promotion trait is a cherished outcome. More attention is being paid to strains endophytic, producing volatile organic compounds, or to the identification of antibiotics and metabolites responsible for the biocontrol. Bioformulation is a critical point in the use of biocontrol agents, but it has been still poorly considered in the experiments. The biocontrol efficacy of well-designed streptomycete consortia or the so-called microbial synthetic communities, possibly in proper bioformulations, and the role of streptomycetes in the reduction of mycotoxins in the grains are aspects that would merit further investigation in the future.

Use of biocontrol agents and botanicals in integrated management of Botrytis cinerea in table grape vineyards.

There is increasing interest in the use of biological control agents (BCAs) and botanicals (BOTs) due to increasing awareness of the environmental and human health risks associated with synthetic plant protection products. The BCAs Bacillus subtilis strain QST713, Bacillus amyloliquefaciens strain D747 and Aureobasidium pullulans strains DSM14940 and DSM14941, and the BOTs Melaleuca alternifolia and terpenic extracts are proposed for the control of grey mould in vineyards. This study was aimed at evaluating their effectiveness in integrated crop management strategies and their outcomes in terms of the management of fungicide resistance and residues. In field trials carried out on table grapes in southern Italy, use of BCAs or BOTs alternately or mixtures of BCAs or BOTs with the succinate dehydrogenase inhibitor fungicide fluopyram showed efficacy of up to 96% against grey mould on bunches, comparable with the chemical reference strategy (up to 87%). By contrast, use of BCAs or BOTs (up to 11 sprays) alone was not effective (< 30%) under high disease pressure. The integrated use of BCAs or BOTs reduced the spread of succinate dehydrogenase inhibitor-resistant conidia, as well as fungicide residues in grapes. Spray schedules based on integration of BCAs or BOTs with fungicides are effective against grey mould and reduce the risk of fungicide resistance in B. cinerea and fungicide residues in grapes. © 2017 Society of Chemical Industry.

Hiperparasitism on mycotoxigenic fungus Aspergillusochraceus G. Wilh. By Cladosporiumcladosporioides (Fresen) de Vries

Biocontrol agents (BCAs) for the control of fungal diseases and pests has been the focus in the development of, thus, the agrochemical industry has refocused priorities on integrated control to include the use of BCAs in research and programmes for disease and pest control. The mechanism of action has often included direct antagonism between the BCA and fungal pathogen, competitive exclusion of the pathogen by niche occupation, production of secondary metabolites, hyperparasitism, and the production of volatile organic compounds (VOCs). It is considered as a characteristic of antagonist, to present more than one mechanism, because in this way, their chances of success are increased. The present study, objectived to evaluate, in vitro, competition between the fungus considered a bioprotective agent of the coffee quality Cladosporiumcladosporioides (Fresen) de Vries and the potentially toxigenic fungus Aspergillus ochraceus G. Wilh. Competition between isolates of C. cladosporioides and A. ochraceus was evaluated in vitro conditions by adding conidia aliquotas in Petri dishes containing PDA (potato, dextrose, agar) culture medium.The antagonist relationship between isolates was observed in Olympus SZX7TR-ILA trinocular stereoscopic microscope coupled to a camera and in scanning electron microscopy ZEISS®. The fungus C. cladosporioides (Fresen) de Vries, showed its antagonism on potencialmycotoxigenic fungus A. ochraceus by of antibiosis and hyperparasitism mechanisms.

Environmental Bacteriophages of the Emerging Enterobacterial Phytopathogen, Dickeya solani, Show Genomic Conservation and Capacity for Horizontal Gene Transfer between Their Bacterial Hosts.

Dickeya solani is an economically important phytopathogen widespread in mainland Europe that can reduce potato crop yields by 25%. There are no effective environmentally-acceptable chemical systems available for diseases caused by Dickeya. Bacteriophages have been suggested for use in biocontrol of this pathogen in the field, and limited field trials have been conducted. To date only a small number of bacteriophages capable of infecting D. solani have been isolated and characterized, and so there is a need to expand the repertoire of phages that may have potential utility in phage therapy strategies. Here we describe 67 bacteriophages from environmental sources, the majority of which are members of the viral family Myoviridae. Full genomic sequencing of two isolates revealed a high degree of DNA identity with D. solani bacteriophages isolated in Europe in the past 5 years, suggesting a wide ecological distribution of this phage family. Transduction experiments showed that the majority of the new environmental bacteriophages are capable of facilitating efficient horizontal gene transfer. The possible risk of unintentional transfer of virulence or antibiotic resistance genes between hosts susceptible to transducing phages cautions against their environmental use for biocontrol, until specific phages are fully tested for transduction capabilities.