Behavior and interactions of the plant growth-promoting bacteria Azospirillum oryzae NBT506 and Bacillus velezensis UTB96 in a co-culture system.

The objective of the present study was to evaluate possible interactions between two potential plant growth-promoting bacteria (PGPB): Azospirillum oryzae strain NBT506 and Bacillus velezensis strain UTB96. To do this, the growth kinetic, biofilm formation, motility, surfactin production, indole-3-acetic acid (IAA) production, phosphate solubilization and enzyme activities of the strains were measured in monoculture and co-culture. The maximum biomass production for the strains in monoculture and co-culture was about 1011 CFU/ml, confirming that these two strains have the potential to grow in co-culture without reduction of biomass efficiency. The co-culture system showed more stable biofilm formation until the end of day 3. Azospirillum showed the maximum IAA production (41.5 mg/l) in a monoculture compared to other treatments. Surfactin promoted both swimming and swarming motility in all treatments. The Bacillus strain in the monoculture and co-culture showed high phosphate solubilizing capability, which increased continuously in the co-culture system after 6 days. The strains showed protease, amylase and cellulase activities in both monoculture and co-culture forms. Chitinase and lipase activities were observed in both the monoculture of the Bacillus strain and the co-culture. Overall, our findings highlight the promotion of biological and beneficial effects of these bacteria when growing together in co-culture.

Responses of cucumber (Cucumis sativus L.) rhizosphere microbial community to some agronomic management practices.

The microbial communities associated to the rhizosphere (the rhizomicrobiome) have a substantial impact on plant growth and yield. Understanding the effects of agricultural management on the rhizomicrobiome is very important for selecting efficient practices. By sequencing the V4 region of 16S rRNA for bacteria and the ITS1 regions and fungi, we investigated the influences of agronomic practices, including cucumber grafting on cucurbit hybrid (Cucurbita moschata × C. maxima), cucumber-garlic intercropping, and treatment with fungicide iprodione-carbendazim on cucumber rhizosphere microbial communities during plant growth. Soil dehydrogenase activity (DHA) and plant vegetative parameters were assessed as an indicator of overall soil microbial activity. We found that both treatments and growth stage induced significant shifts in microbial community structure. Grafting had the highest number of differentially abundant OTUs compared to control samples, followed by intercropping and fungicide treatment, while plant development stage affected both alpha and beta diversities indices and composition of the rhizomicrobiome. DHA was more dependent on plant growth stages than on treatments. Among the assessed factors, grafting and plant developmental stage resulted in the greatest changes in the microbial community composition. Grafting also increased the plant growth parameters, suggesting that this method should be further investigated in vegetable production systems.

Bacillus velezensis UTB96 Is an Antifungal Soil Isolate with a Reduced Genome Size Compared to That of Bacillus velezensis FZB42.

Bacillus velezensis UTB96 was isolated from soil based on its antifungal activity. Whole-genome sequencing of strain UTB96 provided further information about its secondary metabolite gene clusters. Compared to the well-known strain FZB42, UTB96 lacks an IS3 element and a type I restriction endonuclease.

Biodegradation and ecotoxicological impact of cellulose nanocomposites in municipal solid waste composting.

Biodegradable nanocomposites were prepared from polyvinyl alcohol (PVA) and cellulose nanofiber (CNF) by using liquid nitrogen, freeze drying and hot press techniques. The effect of CNF content on the biodegradability of the films was investigated by visual observation, scanning electron microscopy (SEM), weight loss, CO2 evolution, differential scanning calorimetry, measuring the amount of mineralized carbon of the specimens buried in municipal solid waste. Ecotoxicity was evaluated by plants growth tests with cress and spinach. The results confirmed that the weight loss of nanocomposites was lower than that of neat PVA because of the zigzag pathways of microorganisms in the CNF presence. The SEM analysis showed extensive surface roughness and cracks for all samples, indicating the initiation of biodegradation. The CO2 evolution decreased with increasing CNF content from 0% to 10% and then, increased with further increase in the filler content (up to 30 wt%). The crystallinity of the PVA and its nanocomposites increased as a function of time because of the amorphous parts degradation. Preliminary results of the ecotoxicological test revealed that all the nanocomposites and neat PVA did not generate any negative effects on germination or development of the studied vegetal species.

Detection of phlD gene in some fluorescent pseudomonads isolated from Iran and its relative with antifungal activities.

Fluorescent pseudomonads that produce antibiotic 2,4-diacetylphloroglocinol (2,4-DAPG) are important group of PGRP that inhibit a broad spectrum of plant pathogenic fungi. Studying on genetic diversity of 2,4-diacetylphloroglucinol-producing fluorescent pseudomonads has been shown with special importance. The first step to investigate the genetic diversity of these bacteria is detecting of the genes required for the biosynthesis of this antibiotic. The objectives of the current study were detection of phlD gene within fluorescent pseudomonads by a PCR-based assay, and comparison of phenotypic and genotypic characteristics of fluorescent pseudomonads with proven biocontrol potential against some soil-borne phytopathogenic fungi. We used a collection of 47 fluorescent Pseudomonas spp. some with known biological control activity against Macrophomina phaseolina, Rhizoctonia solani, Phytophthora nicotianae var. parasitica, Pythium sp. and Fusarium sp. in vitro and the potential to produce known secondary metabolites such as, siderophore, HCN and protease. The results indicated that 66, 40.42, 63.82,48.94 and 27.65% of strains revealed antagonistic activity against R. solani, M. phaseolina, Pythium sp., P. nicotianae and Fusarium sp., respectively. Rhizoctonia solani recognized as the most vulnerable fungus. Among 47 strains, 76.59, 97.87 and 17% of strains produced protease, siderophore and HCN, respectively. We could detect phlD gene in strains P-5, P-32, P-47. Strain CHA0 was used as positive control for the detection this gene. Overall, there was no obvious link between the existence of phlD gene and inhibition of fungal growth or production of the antifungal metabolites in vitro. But in some strains such as CHA0 and P-5, we saw a link between the existence of phlD and antifungal activities. Studying on detection and diversity of phlD provides a fundamental knowledge for developing a rapid genetic screening system to identify a potential biocontrol strains.

Diazinon dissipation in pesticide-contaminated paddy soil: kinetic modeling and isolation of a degrading mixed bacterial culture.

Dissipation kinetics of diazinon was investigated in soils culled from a paddy field with a long history of the pesticide application. Goodness of fit statistical indices derived from several fitted mono- and bi-exponential kinetic models revealed a bi-phasic pattern of the diazinon dissipation curve at 15 and 150 mg kg-1 spiking levels, which could be described best by the first-order double exponential decay (FODED) model. Parameters obtained from this model were able to describe the enhanced dissipation of diazinon as the result of repeated soil applications, where a larger fraction of the pesticide readily available in the solution phase was dissipated with a fast rate. Cluster and principal component analysis (PCA) of denaturing gradient gel electrophoresis (DGGE) obtained from soil bacterial populations revealed that they were only affected at the 150 mg kg-1 diazinon concentration. This was also supported by the phylogenetic tree obtained from sequences of the main gel bands. Accordingly, bacterial populations belonging to Proteobacteria were enriched in the soil following three treatments with diazinon at 150 mg kg-1. The Shannon's index revealed a nonsignificant increase (P ≤ 0.05) in overall diversity of soil bacteria following diazinon application. Diazinon-degrading bacteria were isolated from the paddy soils in a mineral salt medium. Results showed that the isolated mixed culture was able to remove 90% of the pesticide at two concentrations of 50 and 100 mg L-1 by 16.81 and 19.60 days, respectively. Sequencing the DGGE bands confirmed the role of Betaproteobacteria as the main components of the isolated mixed culture in the degradation of diazinon.

Comparative Analyses of Transport Proteins Encoded within the Genomes of Bdellovibrio bacteriovorus HD100 and Bdellovibrio exovorus JSS.

Bdellovibrio, δ-proteobacteria, including B. bacteriovorus (Bba) and B. exovorus (Bex), are obligate predators of other Gram-negative bacteria. While Bba grows in the periplasm of the prey cell, Bex grows externally. We have analyzed and compared the transport proteins of these 2 organisms based on the current contents of the Transporter Classification Database (TCDB; www.tcdb.org). Bba has 103 transporters more than Bex, 50% more secondary carriers, and 3 times as many MFS carriers. Bba has far more metabolite transporters than Bex as expected from its larger genome, but there are 2 times more carbohydrate uptake and drug efflux systems, and 3 times more lipid transporters. Bba also has polyamine and carboxylate transporters lacking in Bex. Bba has more than twice as many members of the Mot-Exb family of energizers, but both may have energizers for gliding motility. They use entirely different types of systems for iron acquisition. Both contain unexpectedly large numbers of pseudogenes and incomplete systems, suggesting that they are undergoing genome size reduction. Interestingly, all 5 outer-membrane receptors in Bba are lacking in Bex. The 2 organisms have similar numbers and types of peptide and amino acid uptake systems as well as protein and carbohydrate secretion systems. The differences observed correlate with and may account, in part, for the different lifestyles of these 2 bacterial predators.

Fusarium culmorum affects expression of biofilm formation key genes in Bacillus subtilis.

It is known that there is correlation between biofilm formation and antagonistic activities of Bacillus subtilis strains; but, the mechanism of this correlation is not clear. So, the effect of the plant pathogen (Fusarium culmorum) on the biofilm formation in a B. subtilis strain with high antagonistic and biofilm formation activities was studied. The expression of sinR and tasA genes involved in the biofilm formation was studied in both single culture of bacterium (B) and co-culture with F. culmorum (FB) using real-time PCR. The results revealed that the expression of the sinR gene in both B and FB conditions was continuously decreased during the biofilm formation period and, after 24h (B4 and FB4), it reached 1% and 0.3% at the planktonic phase (B1), respectively, whereas the expression of the tasA was continuously increased and was 5.27 and 30 times more than that at the planktonic phase (B1) after 24h, respectively. So, the expression reduction rate for sinR (3 times) and the expression increasing rate for tasA (6 times) were significantly higher in FB conditions than the B ones. The relative expression of sinR in FB1 (planktonic phase), FB2 (8h), FB3(12h), and FB4 (24h) times was 0.65, 0.44, 0.35, and 0.29, whereas the tasA gene expression was 2.98, 3.44, 4.37, and 5.63-fold of the one at coordinate time points in B conditions, respectively. The significant expression reduction of sinR and increase of tasA confirmed that the presence of pathogen could stimulate biofilm formation in the antagonistic bacterium.

Inhibition of the Aspergillus flavus Growth and Aflatoxin B1 Contamination on Pistachio Nut by Fengycin and Surfactin-Producing Bacillus subtilis UTBSP1.

In this study, the treatment of pistachio nuts by Bacillus subtilis UTBSP1, a promising isolate to degrade aflatoxin B1 (AFB1), caused to reduce the growth of Aspergillus flavus R5 and AFB1 content on pistachio nuts. Fluorescence probes revealed that the cell free supernatant fluid from UTBSP1 affects spore viability considerably. Using high-performance liquid chromatographic (HPLC) method, 10 fractions were separated and collected from methanol extract of cell free supernatant fluid. Two fractions showed inhibition zones against A. flavus. Mass spectrometric analysis of the both antifungal fractions revealed a high similarity between these anti-A. flavus compounds and cyclic-lipopeptides of surfactin, and fengycin families. Coproduction of surfactin and fengycin acted in a synergistic manner and consequently caused a strong antifungal activity against A. flavus R5. There was a positive significant correlation between the reduction of A. flavus growth and the reduction of AFB1 contamination on pistachio nut by UTBSP1. The results indicated that fengycin and surfactin-producing B. subtilis UTBSP1 can potentially reduce A. flavus growth and AFB1 content in pistachio nut.

Fusarium culmorum affects expression of biofilm formation key genes in Bacillus subtilis

Abstract It is known that there is correlation between biofilm formation and antagonistic activities of Bacillus subtilis strains; but, the mechanism of this correlation is not clear. So, the effect of the plant pathogen (Fusarium culmorum) on the biofilm formation in a B. subtilis strain with high antagonistic and biofilm formation activities was studied. The expression of sinR and tasA genes involved in the biofilm formation was studied in both single culture of bacterium (B) and co-culture with F. culmorum (FB) using real-time PCR. The results revealed that the expression of the sinR gene in both B and FB conditions was continuously decreased during the biofilm formation period and, after 24 h (B4 and FB4), it reached 1% and 0.3% at the planktonic phase (B1), respectively, whereas the expression of the tasA was continuously increased and was 5.27 and 30 times more than that at the planktonic phase (B1) after 24 h, respectively. So, the expression reduction rate for sinR (3 times) and the expression increasing rate for tasA (6 times) were significantly higher in FB conditions than the B ones. The relative expression of sinR in FB1 (planktonic phase), FB2 (8 h), FB3(12 h), and FB4 (24 h) times was 0.65, 0.44, 0.35, and 0.29, whereas the tasA gene expression was 2.98, 3.44, 4.37, and 5.63-fold of the one at coordinate time points in B conditions, respectively. The significant expression reduction of sinR and increase of tasA confirmed that the presence of pathogen could stimulate biofilm formation in the antagonistic bacterium.