Effects of different light wavelengths on Bacillus subtilis and Bacillus velezensis, two biocontrol agents isolated from the maize phyllosphere.

In previous studies, two strains isolated from the maize phyllosphere were identified as Bacillus subtilis (EM-A7) and Bacillus velezensis (EM-A8) and selected as potential biocontrol agents against Exserohilum turcicum. This study aimed to assess the ability of EM-A7 and EM-A8 to form biofilm and have antagonistic activity under varying light conditions. LED sources were custom-designed so that each corresponded to a given spectrum at a specific photosynthetically active photon flux density. Significant differences were observed in growth parameters (generation time and constant growth rate) under different LED sources. Blue light inhibited the growth of both strains. Red increased k rate in EM-A8, while the g values increased in EM-A7. Red and white light generally increased biofilm formation, and blue light inhibited it. EM-A7 and EM-A8 significantly reduced their ability to swim under blue LED, but it was not affected by red, green, or white light. The ability to swarm was negatively affected. Fungal growth decreased significantly compared to the control when the bacterium growing on the same plate had been previously incubated under red and white light or in the dark. These results indicate that different light wavelengths clearly influenced the aspects assessed in B. subtilis and B. velezensis, with the effects of blue light being overall negative and those of red and white overall positive. Given that, all these factors can be important for the establishment and survival of Bacillus strains on leaves, as well as for their effectiveness against pathogens, light could be a significant factor to consider in the design of biocontrol strategies.

Could Bacillus biofilms enhance the effectivity of biocontrol strategies in the phyllosphere?

Maize (Zea mays L.), a major crop in Argentina and a staple food around the world, is affected by the emergence and re-emergence of foliar diseases. Agrochemicals are the main control strategy nowadays; however, they can cause resistance in insects and microbial pathogens and have negative effects on the environment and human health. An emerging alternative is the use of living organisms, i.e. microbial biocontrol agents, to suppress plant pathogen populations. This is a risk-free approach when the organisms acting as biocontrol agents come from the same ecosystem as the foliar pathogens they are meant to antagonize. Some epiphytic microorganisms may form biofilm by becoming aggregated and attached to a surface, as is the case of spore-forming bacteria from the genus Bacillus. Their ability to sporulate and their tolerance to long storage periods make them a frequently used biocontrol agent. Moreover, the biofilm that they create protects them against different abiotic and biotic factors and helps them to acquire nutrients, which ensures their survival on the plants they protect. This review analyzes the interactions that the phyllosphere-inhabiting Bacillus genus establishes with its environment through biofilm, and how this lifestyle could serve to design effective biological control strategies.

Sexuality generates diversity in the aflatoxin gene cluster: evidence on a global scale.

Aflatoxins are produced by Aspergillus flavus and A. parasiticus in oil-rich seed and grain crops and are a serious problem in agriculture, with aflatoxin B1 being the most carcinogenic natural compound known. Sexual reproduction in these species occurs between individuals belonging to different vegetative compatibility groups (VCGs). We examined natural genetic variation in 758 isolates of A. flavus, A. parasiticus and A. minisclerotigenes sampled from single peanut fields in the United States (Georgia), Africa (Benin), Argentina (Córdoba), Australia (Queensland) and India (Karnataka). Analysis of DNA sequence variation across multiple intergenic regions in the aflatoxin gene clusters of A. flavus, A. parasiticus and A. minisclerotigenes revealed significant linkage disequilibrium (LD) organized into distinct blocks that are conserved across different localities, suggesting that genetic recombination is nonrandom and a global occurrence. To assess the contributions of asexual and sexual reproduction to fixation and maintenance of toxin chemotype diversity in populations from each locality/species, we tested the null hypothesis of an equal number of MAT1-1 and MAT1-2 mating-type individuals, which is indicative of a sexually recombining population. All samples were clone-corrected using multi-locus sequence typing which associates closely with VCG. For both A. flavus and A. parasiticus, when the proportions of MAT1-1 and MAT1-2 were significantly different, there was more extensive LD in the aflatoxin cluster and populations were fixed for specific toxin chemotype classes, either the non-aflatoxigenic class in A. flavus or the B1-dominant and G1-dominant classes in A. parasiticus. A mating type ratio close to 1∶1 in A. flavus, A. parasiticus and A. minisclerotigenes was associated with higher recombination rates in the aflatoxin cluster and less pronounced chemotype differences in populations. This work shows that the reproductive nature of the population (more sexual versus more asexual) is predictive of aflatoxin chemotype diversity in these agriculturally important fungi.

Fusarium poae Pathogenicity and Mycotoxin Accumulation on Selected Wheat and Barley Genotypes at a Single Location in Argentina.

Fusarium poae is a relatively weak pathogen with increasing importance in cereal grains, principally due to its capacity to produce several mycotoxins. In this study, we evaluated the pathogenicity and toxin accumulation of individual F. poae isolates on wheat and barley under natural conditions for 3 years. Analysis of variance demonstrated significant differences for year-genotype, year-isolate, genotype-isolate, and year-genotype-isolate interactions for both incidence and disease severity. Based on contrast analysis, 'Apogee' was more susceptible than the other wheat genotypes, wheat genotypes were more susceptible than barley genotypes, durum wheat genotypes were more susceptible than bread wheat genotypes, and barley genotype 'Scarlett' had greater symptom development per spike than the other barley genotypes. Neither HT-2 nor T-2 toxins were detected in the grain samples. However, high levels of nivalenol were found in both wheat and barley samples. The increased reported isolation of F. poae from wheat and barley and the high capacity of this fungus to produce nivalenol underlie the need for more studies on F. poae-host interactions, especially for barley.

Physiological Effects of Microbial Biocontrol Agents in the Maize Phyllosphere.

In a world with constant population growth, and in the context of climate change, the need to supply the demand of safe crops has stimulated an interest in ecological products that can increase agricultural productivity. This implies the use of beneficial organisms and natural products to improve crop performance and control pests and diseases, replacing chemical compounds that can affect the environment and human health. Microbial biological control agents (MBCAs) interact with pathogens directly or by inducing a physiological state of resistance in the plant. This involves several mechanisms, like interference with phytohormone pathways and priming defensive compounds. In Argentina, one of the world's main maize exporters, yield is restricted by several limitations, including foliar diseases such as common rust and northern corn leaf blight (NCLB). Here, we discuss the impact of pathogen infection on important food crops and MBCA interactions with the plant's immune system, and its biochemical indicators such as phytohormones, reactive oxygen species, phenolic compounds and lytic enzymes, focused mainly on the maize-NCLB pathosystem. MBCA could be integrated into disease management as a mechanism to improve the plant's inducible defences against foliar diseases. However, there is still much to elucidate regarding plant responses when exposed to hemibiotrophic pathogens.

Impact of cycling temperatures on Fusarium verticillioides and Fusarium graminearum growth and mycotoxins production in soybean.

BACKGROUND: Fusarium graminearum and F. verticillioides are two very important mycotoxigenic species as they cause diverse diseases in crops. The effects of constant and cycling temperatures on growth and mycotoxin production of these species were studied on soybean based medium and on irradiated soya beans. RESULTS: F. graminearum grew better when was incubated at 15, 20 and 15-20 °C (isothermal or cycling temperature) during 21 days of incubation. Maximum levels of zearalenone and deoxynivalenol (39.25 and 1040.4 µg g(-1), respectively) were detected on soya beans after 15 days of incubation and the optimal temperature for mycotoxin production was 15 °C for zearalenone and 20 °C for deoxynivalenol. F. verticillioides grew better at 25 °C in culture medium and at 15/20 °C and 15/25 °C on soybean seeds. Fumonisin B(1) was produced only in culture medium, and the maximum level (7.38 µg g(-1)) was found at 15 °C after 7 days of incubation. CONCLUSION: When growth and mycotoxin production under cycling temperatures were predicted from the results under constant conditions, observed values were different from calculated for both species and substrate medium. Therefore, care should be taken if data at constant temperature conditions are to be extrapolated to real field conditions.

In vitro studies of biofilm-forming Bacillus strains, biocontrol agents isolated from the maize phyllosphere.

We aimed to assess how biofilm formation by three Bacillus isolates was affected by changes in temperature, water potential, growth media, time, and the combinations between these factors. The strains had been selected as potential biological control agents (BCAs) in earlier studies, and they were identified as B. subtilis and B. velezensis spp. through 16 rRNA sequencing and MALDI-TOF MS. Maize leaves (ML) were used as one of the growth media, since they made it possible to simulate the nutrient content in the maize phyllosphere, from which the bacteria were originally isolated. The strains were able to form biofilm both in ML and biofilm-inducing MSgg after 24, 48, and 72 h. Biofilm development in the form of pellicles and architecturally complex colonies varied morphologically from one strain to another and depended on the conditions mentioned above. In all cases, colonies and pellicles were less complex when both temperature and water potential were lower. Scanning electron microscopy (SEM) revealed that changing levels of complexity in pellicles were correlated with those in colonies. Statistical analyses found that the quantification of biofilm produced by the isolates was influenced by all the conditions tested. In terms of motility (which may contribute to biofilm formation), swimming and swarming were possible for all strains in 0.3 and 0.7% agar, respectively. A more in-depth understanding of how abiotic factors influence biofilm formation can contribute to a more effective use of these biocontrol strains against pathogens in the maize phyllosphere.

Improvement of simultaneous Cr(VI) and phenol removal by an immobilised bacterial consortium and characterisation of biodegradation products.

Microbial bioremediation emerged some decades ago as an eco-friendly technology to restore polluted sites. Traditionally, the search for microorganisms suitable for bioremediation has been based on the selection of isolated strains able to remove a specific type of pollutant. However, this strategy has now become obsolete, since co-pollution is a global reality. Thus, current studies attempt to find bacterial cultures capable of coping with a mixture of organic and inorganic compounds. In this sense, the bacterial consortium SFC 500-1 has demonstrated efficiency for Cr(VI) and phenol removal, both of which are found in many industrial wastewaters. In the present study, the ability of SFC 500-1 for simultaneous removal was improved through its entrapment in a Ca-alginate matrix. This strategy led to an increased removal of Cr(VI), which was partially reduced to Cr(III). Immobilised cells were able to tolerate and degrade phenol up to 1,500mg/l at high rates, forming catechol and cis,cis-muconate as oxidation intermediates. Successful removal potential through 5 cycles of reuse, as well as after long-term storage, was another important advantage of the immobilised consortium. These characteristics make SFC 500-1 an interesting system for potential application in the biotreatment of co-polluted effluents.

Growth promotion of peanut (Arachis hypogaea L.) and maize (Zea mays L.) plants by single and mixed cultures of efficient phosphate solubilizing bacteria that are tolerant to abiotic stress and pesticides.

The aims of this study were, to analyze in vitro phosphate solubilization activity of six native peanut bacteria and to determine the effect of single and mixed inoculation of these bacteria on peanut and maize plants. Ability to produce organic acids and cofactor PQQ, to solubilize FePO4 and AlPO4 and phosphatase activity were analyzed. Also, the ability to solubilize phosphate under abiotic stress and in the presence of pesticides of the selected bacteria was determined. The effect of single and mixed bacterial inocula was analyzed on seed germination, maize plant growth and in a crop rotation plant assay with peanut and maize. The six strains produced gluconic acid and five released cofactor PQQ into the medium. All bacteria showed ability to solubilize phosphate from FePO4 and AlPO4 and phosphatase activity. The ability of the bacteria to solubilize tricalcium phosphate under abiotic stress and in presence of pesticides indicated encouraging results. Bacterial inoculation on peanut and maize increased seed germination, plant́s growth and P content. Phosphate solubilizing bacteria used in this study showed efficient phosphate mineralizing and solubilization ability and would be potential P-biofertilizers for peanut and maize.

Non-aflatoxigenic Aspergillus flavus as potential biocontrol agents to reduce aflatoxin contamination in peanuts harvested in Northern Argentina.

Biological control is one of the most promising strategies for preventing aflatoxin contamination in peanuts at field stage. A population of 46 native Aspergillus flavus nonaflatoxin producers were analysed based on phenotypic, physiological and genetic characteristics. Thirty-three isolates were characterized as L strain morphotype, 3 isolates as S strain morphotype, and 10 isolates did not produce sclerotia. Only 11 of 46 non-aflatoxigenic isolates did not produce cyclopiazonic acid. The vegetative compatibility group (VCG) diversity index for the population was 0.37. For field trials we selected the non-aflatoxigenic A. flavus AR27, AR100G and AFCHG2 strains. The efficacy of single and mixed inocula as potential biocontrol agents in Northern Argentina was evaluated through a 2-year study (2014-2015). During the 2014 peanut growing season, most of the treatments reduced the incidence of aflatoxigenic strains in both soil and peanut kernel samples, and no aflatoxin was detected in kernels. During the 2015 growing season, there was a reduction of aflatoxigenic strains in kernel samples from the plots treated with the potential biocontrol agents. Reductions of aflatoxin contamination between 78.36% and 89.55% were observed in treated plots in comparison with the un-inoculated control plots. This study provides the first data on aflatoxin biocontrol based on competitive exclusion in the peanut growing region of Northern Argentina, and proposes bioproducts with potential use as biocontrol agents.

Development of a PCR assay to detect the potential production of nivalenol in Fusarium poae.

Fusarium species can produce mycotoxins, which can contaminate cereal-based food producing adverse effects for human and animal health. In recent years, the importance of Fusarium poae has increased within the Fusarium head blight complex. Fusarium poae is known to produce trichothecenes, especially nivalenol, a potent mycotoxin able to cause a variety of toxic effects. In this study, a specific primer pair was designed based on the tri7 gene to detect potential nivalenol-producing F. poae isolates. A total of 125 F. poae, four F. cerealis, two F. culmorum, one F. langsethiae, one F. sporotrichioides and seven F. graminearum, plus F. austroamericanum, F. meridionale, F. graminearum sensu stricto and F. cortaderiae from the NRRL collection were analysed, and only F. poae isolates gave a positive result for the presence of a 296-bp partial tri7 DNA fragment. Moreover, the primer set was tested from cereal seed samples where F. poae and other Fusarium species with a negative result for the specific reaction ( F. graminearum, F. oxysporum, F. chlamydosporum, F. sporotrichioides, F. equiseti and F. acuminatum) were isolated, and the expected fragment was amplified. We developed a rapid and reliable PCR assay to detect potential nivalenol-producing F. poae isolates.

Influence of water activity and temperature on growth and mycotoxin production by Alternaria alternata on irradiated soya beans.

The aim of this study was to determine the effects of water activity (a(w)) (0.99-0.90), temperature (15, 25 and 30°C) and their interactions on growth and alternariol (AOH) and alternariol monomethyl ether (AME) production by Alternaria alternata on irradiated soya beans. Maximum growth rates were obtained at 0.980 a(w) and 25°C. Minimum a(w) level for growth was dependent on temperature. Both strains were able to grow at the lowest a(w) assayed (0.90). Maximum amount of AOH was produced at 0.98 a(w) but at different temperatures, 15 and 25°C, for the strains RC 21 and RC 39 respectively. Maximum AME production was obtained at 0.98 a(w) and 30°C for both strains. The concentration range of both toxins varied considerably depending on a(w) and temperature interactions. The two metabolites were produced over the temperature range 15 to 30°C and a(w) range 0.99 to 0.96. The limiting a(w) for detectable mycotoxin production is slightly greater than that for growth. Two-dimensional profiles of a(w)× temperature were developed from these data to identify areas where conditions indicate a significant risk from AOH and AME accumulation on soya bean. Knowledge of AOH and AME production under marginal or sub-optimal temperature and a(w) conditions for growth can be important since improper storage conditions accompanied by elevated temperature and moisture content in the grain can favour further mycotoxin production and lead to reduction in grain quality. This could present a hazard if the grain is used for human consumption or animal feedstuff.