Pomegranate Rhizosphere Microbial Diversity Revealed by Metagenomics: Toward Organic Farming, Plant Growth Promotion and Biocontrol?

Food production must undergo systems change to meet the sustainable development goals (SDGs). For example, organic farming can be empowered by soil microorganisms with plant growth promotion (PGP) and biocontrol features. In this context, there have been limited studies on pomegranate. We investigated microbial diversity in rhizosphere of the pomegranate "Bhagwa" variety and its potential role in PGP and biocontrol. Both bulk and rhizosphere soil samples were analyzed for their physicochemical properties. Whole metagenome sequencing was conducted using the Illumina NovaSeq6000 platform. Surprisingly, we found that bulk and rhizosphere soil samples had comparable microbial diversity. Metagenome sequencing revealed the abundance of Streptomyces indicus, Bradyrhizobium kalamazoonesis, and Pseudomonas cellulosum in the rhizosphere that are reported here for the first time in agricultural literature. Pathway prediction analysis using KEGG (Kyoto Encyclopedia for Genes and Genomes) and COG (clusters of orthologous genes) databases identified metabolic pathways associated with biocontrol properties against pathogens. We confirmed the metagenome data in vitro, which demonstrated their PGP potential and antimicrobial properties. For instance, S. indicus produced high concentration of indole-3-acetic acid, a PGP phytohormone, that can stimulate plant growth. In addition, an antimicrobial susceptibility assay suggested that bacterial extracts displayed activity against Xanthomonas, a primary pathogen causing the pomegranate wilt disease. In conclusion, this study suggests that S. indicus, B. kalamazoonesis, and P. cellulosum can potentially be PGP and biocontrol agents that may contribute to increased crop productivity in pomegranate cultivation. These agents and their combinations warrant future research with an eye on SDGs and so as to enable and innovate organic farming and pomegranate agricultural practices.

Cadmium/zinc stresses and plant cultivation influenced soil microflora: a pot experiment conducted in field.

It's of great challenge to address for heavy metal-contaminated soil. Once the farmland is contaminated with heavy metals, the microbial ecology of the plant rhizosphere will change, which in turn impacts crop productivity and quality. However, few studies have explored the effects of heavy metals on plant rhizosphere microbes in farmland and the role that plant cultivation plays in such a phytoremediation practice. In this study, the impacts of comfrey (Symphytum officinale L.) cultivation and the stresses of cadmium/zinc (Cd/Zn) on rhizosphere soil microflora were examined. Microbial DNA was collected from soils to evaluate the prevalence of bacteria and fungi communities in rhizosphere soils. High-throughput 16 S rRNA sequencing was used to determine the diversity of the bacterial and fungal communities. The results showed that growing comfrey on polluted soils reduced the levels of Cd and Zn from the vertical profile. Both the comfrey growth and Cd/Zn stresses affected the community of rhizosphere microorganisms (bacteria or fungi). Additionally, the analysis of PCoA and NMDS indicated that the cultivation of comfrey significantly changed the bacterial composition and structure of unpolluted soil. Comfrey cultivation in polluted and unpolluted soils did not result in much variance in the fungi's species composition, but the fungal compositions of the two-type soils were noticeably different. This work provided a better understanding of the impacts of Cd/Zn stresses and comfrey cultivation on rhizosphere microbial community, as well as new insight into phytoremediation of heavy metal-contaminated soils.

Regulating pathway for bacterial diversities toward improved ecological benefits of thiencarbazone-methyl·isoxaflutole application: A field experiment.

Herbicide abuse has a significantly negative impact on soil microflora and further influences the ecological benefit. The regulating measures and corresponding mechanisms mitigating the decreased bacterial diversity due to herbicide use have rarely been studied. A field experiment containing the application gradient of an efficient maize herbicide thiencarbazone-methyl·isoxaflutole was performed. The relationship between soil bacterial community and thiencarbazone-methyl·isoxaflutole use was revealed. Modified attapulgite was added to explore its impacts on soil microflora under the thiencarbazone-methyl·isoxaflutole application. Based on the analytic network process-entropy weighting method-TOPSIS method model, the ecological benefit focusing on microbial responses was quantitatively estimated along with technical effectiveness and economic benefit. The results showed that the diversity indices of soil microflora, especially the Inv_Simpson index, were reduced at the recommended, 5 and 10 times the recommended dosages of thiencarbazone-methyl·isoxaflutole use. The Flavisolibacter bacteria was negatively correlated with the residues in soils based on the random forest model and correlation analysis, indicating a potential degrader of thiencarbazone-methyl·isoxaflutole residues. The structural equation model further confirmed that the high soil water content and soil pH promoted the function of Flavisolibacter bacteria, facilitated the dissipation of thiencarbazone-methyl·isoxaflutole residues and further improved the diversity of soil microflora. In addition, the presence of modified attapulgite was found to increase the soil pH, which may improve bacterial diversity through the regulating pathway. This explained the high ecological benefits of the treatment where the thiencarbazone-methyl·isoxaflutole was applied at the recommended dosage rates in conjunction with modified attapulgite addition. Therefore, the comprehensive benefits of thiencarbazone-methyl·isoxaflutole application with a focus on ecological benefits can be improved by regulating the soil pH with modified attapulgite.

A review of the impact of herbicides and insecticides on the microbial communities.

Enhancing crop yield to accommodate the ever-increasing world population has become critical, and diminishing arable land has pressured current agricultural practices. Intensive farming methods have been using more pesticides and insecticides (biocides), culminating in soil deposition, negatively impacting the microbiome. Hence, a deeper understanding of the interaction and impact of pesticides and insecticides on microbial communities is required for the scientific community. This review highlights the recent findings concerning the possible impacts of biocides on various soil microorganisms and their diversity. This review's bibliometric analysis emphasised the recent developments' statistics based on the Scopus document search. Pesticides and insecticides are reported to degrade microbes' structure, cellular processes, and distinct biochemical reactions at cellular and biochemical levels. Several biocides disrupt the relationship between plants and their microbial symbionts, hindering beneficial biological activities that are widely discussed. Most microbial target sites of or receptors are biomolecules, and biocides bind with the receptor through a ligand-based mechanism. The biomarker action mechanism in response to biocides relies on activating the receptor site by specific biochemical interactions. The production of electrophilic or nucleophilic species, free radicals, and redox-reactive agents are the significant factors of biocide's metabolic reaction. Most studies considered for the review reported the negative impact of biocides on the soil microbial community; hence, technological development is required regarding eco-friendly pesticide and insecticide, which has less or no impact on the soil microbial community.

Interaction of TiO2 nanoparticles with soil: Effect on microbiological and chemical traits.

Titanium dioxide (TiO2) nanoparticles (NPs) are the most widely used nanomaterials and their expanding use raises concerns about their impacts on soil ecosystems and functioning. The present study evaluates the potential impacts of TiO2 NPs applied at low doses (0, 1.0, 2.5, 5.0, 10.0 and 20.0 mg L-1) on soil chemical properties including the macro and micronutrient contents, microbial population and enzyme activities in rhizosphere soil of mung bean crop at different time intervals (7, 14, 28 and 56 days). A quantitative RT-PCR study was also performed to study the relative change in the gene expression of ammonia oxidizer and nitrogen fixers upon TiO2 NP supplementation. An increase in soil nutrient content viz., available N, P, Cu, Fe, Mn, nitrate-N and ammonical-N was observed with NP application except available K and Zn content. The TiO2 NPs stimulated the growth of soil microflora at low concentrations while an inhibitory effect was recorded at high concentrations. The soil fungi and actinobacteria emerged as the most sensitive groups of soil microbes towards TiO2 NP exposure exhibiting detrimental impacts on their growth at all concentrations. Similarly, the soil enzyme activities enhanced till TiO2 NPs (10.0 mg L-1) which was followed by decrease at higher concentrations. The qRT-PCR study showed that the ammonia oxidizers were more affected by TiO2 NPs application than nitrogen fixers. These findings suggest that TiO2 NPs can be used as stimulators of soil nutrients and soil microbial dynamics at low concentrations.

Effect of decomposing oil palm trunk fibers on plant growth and soil microbial community composition.

Oil palm trunks (OPT) are logged for replantation and the fiber residues are disposed of into the palm plantation area. The fiber residues are expected to increase soil fertility through recycling of carbon and minerals via fiber decomposition. This study investigated the effects of OPT fiber disposal and other lignocellulosic biomass on plant growth and microbial diversity in the soil environment. Four treatment plots were tested: (A) soil+OPT fiber (1:20), (B) soil+sugarcane bagasse (1:20), (C) soil+cellulose powder (1:20), and (D) unamended soil as a negative control. Low plant height, decreased chlorophyll content, and low biomass was observed in corn grown on soil mixed with OPT fiber, cellulose, and sugarcane bagasse, when compared with those of the control. The plants grown with OPT fiber were deficient in total nitrogen and magnesium when compared with those without fiber amendment, which suggested that nitrogen and minerals in soil might be taken up by changing microflora because of the OPT fibers presence. To confirm differences in the soil microflora, metagenomics analysis was performed on untreated soil and soil from each lignocellulose treatment. The microflora of soils mixed with OPT fiber, cellulose and sugarcane bagasse revealed substantial increases in bacteria such as families Cytophagaceae and Oscillospiraceae, and two major fungal genera, Trichoderma and Trichocladium, that are involved in lignocellulose degradation. OPT fiber resulted in a drastic increase in the ratios and amounts of Trichocladium in the soil when compared with those of cellulose and sugarcane bagasse. These results indicate that unregulated disposal of OPT fiber into plantation areas could result in nutrient loss from soil by increasing the abundance of microorganisms involved in lignocellulose decomposition.

Response of soil enzyme activity and bacterial community to copper hydroxide nanofertilizer and its ionic analogue under single versus repeated applications.

Nanosized agrochemicals like nanofertilizers are being applied to soils. Adverse impacts of nanofertilizers on soil microflora were reported in past studies, but only considering a single application. Repeated applications are however more likely to occur in agriculture. We investigated effects of single versus repeated applications of a copper hydroxide nanofertilizer formulation (NFF) on soil enzyme activity and bacterial community. One or three applications were performed within 21 days to achieve same final level of Cu in soil (48 mg(Cu)/kg: the recommended dose of NFF). Besides, the active ingredient (i.e., copper hydroxide nanotubes (NT)) and dispersing agent (DA) of NFF, and an ionic fertilizer (i.e., CuSO4) were examined. Fluorescein diacetate hydrolase (FDAse), N-acetylglucosaminidase (NAG), leucine aminopeptidase (LAP), and urease (URE) showed negligible changes in the activities between the control and DA treatment. Bacterial community abundance, composition and diversity exhibited similar phenomena. Exposures to copper hydroxide NFF and NT or CuSO4 enhanced the activities of FDAse and NAG, weakened the activity of URE, and showed negligible changes in the LAP activity irrespective of single and repeated applications. Concentrations of NO3--N and NH4+-N in soil were also affected by the application mode of NFF. More importantly, responses of soil bacterial community to copper hydroxide NFF were highly dependent on its application mode, whereas similar responses were observed in the CuSO4 treatment regardless of single or repeated applications. This study provided new insights into environmental risk of copper hydroxide NFF that were ignored in previous studies using a single exposure.

Population density of Trichoderma fungi in natural environments and agrosystems of a Cerrado area / Densidade populacional de fungos do gênero Trichoderma em ambientais naturais e agrossistemas de uma área de Cerrado

Abstract: Soil microorganisms present a great diversity, involving taxonomically distinct groups that play a role in the decomposition of organic matter, nutrient cycling, soil aggregation, among others. In this diversity, the fungi of the genus Trichoderma have been successful plant pathogen biocontrol agents, as plant growth promoters and as inducers of plant resistance to diseases. In addition, they are important in the sustainability of natural ecosystems. Aiming to verify the population density of Trichoderma fungi in natural environments and agroecosystems, in Cerrado area, samples of soils and roots from native vegetation and agroecological production system were collected in the Federal District, Brazil. The collection points were randomly selected, and each soil or root sample was individually wrapped. The soil adhered to the roots was removed for evaluations. Serial sample dilutions and number of Colony Forming Units (CFUs) of Trichoderma isolates were performed. The results showed that the number of CFU varied depending on the plant and location evaluated. The replacement of native vegetation by organic farming systems did not result in a significant reduction in this number.

Resumo: Os microrganismos de solo apresentam uma grande diversidade, envolvendo grupos taxonomicamente distintos que desempenham papel na decomposição da matéria orgânica, ciclagem de nutrientes, agregação dos solos, dentre outros. Nesta diversidade, os fungos do gênero Trichoderma tem apresentado sucesso como agentes de biocontrole de fitopatógenos, como promotores de crescimento de plantas e, ainda, como indutores de resistência de plantas a doenças. Além disso, são importantes na sustentabilidade dos ecossistemas naturais. Com o objetivo de verificar a densidade populacional de fungos do gênero Trichoderma em ambientes naturais e agroecossistemas, em área de Cerrado, amostras de solos e raízes oriundas de vegetação nativa e de sistema de produção agroecológica foram coletadas na região do Distrito Federal, Brasil. Os pontos de coleta foram selecionados aleatoriamente, e cada amostra de solo ou raiz foi acondicionada individualmente. O solo aderido às raízes foi removido para as avaliações. Foram realizadas diluições seriadas das amostras e contagem do número de Unidades Formadoras de Colônias (UFCs) de isolados de Trichoderma. Os resultados mostraram que o número de UFC variou dependendo da planta e da localidade avaliada. A substituição da vegetação nativa por sistemas de cultivo orgânicos não resultou em importante redução neste número.

Influence of chlorothalonil and carbendazim fungicides on the transformation processes of urea nitrogen and related microbial populations in soil.

To improve crop yielding, a large amount of fungicides is continuously applied during the agricultural management, while the effects of fungicides residues on microbial processing of N in soil need further study. In the present study, two broad spectrum fungicides, chlorothalonil and carbendazim, were applied at the rates of 5, 10, and 50 mg of active ingredient (A.I.) per kg of dry soil combined with urea with 200 mg of N per kg of dry soil under laboratory conditions. The results showed that chlorothalonil obviously retarded the hydrolysis of urea, whereas carbendazim accelerated it in 4 days after the treatments (P < 0.05). Chlorothalonil reduced denitrification, nitrification, and N2O production (P < 0.05), but not for carbendazim. Further analysis on N-associated microbial communities showed chlorothalonil reduced nitrosomonas populations at the rates of 10 and 50 mg of A.I. per kg and autotrophic nitrifying bacterial populations at three application rates (P < 0.05), but Carbendazim decreased nitrosomonas populations only at the rate of 50 mg of A.I. per kg and also autotrophic nitrifying bacterial populations at three rates and heterotrophic nitrifying bacterial populations at the rates of 10 and 50 mg of A.I. per kg. The reasons for this difference were ascribed to arrest urea hydrolysis and impediment of denitrification and nitrification processes by chlorothalonil. In conclusion, to improve crop yielding, chlorothalonil might be more beneficial to conserve soil N by improving soil N fertility, compared with carbendazim.

Influence of triazole pesticides on tillage soil microbial populations and metabolic changes.

Pesticides are generally applied to agricultural soil to control crop diseases, with the critical goal of preventing yield loss and optimizing economic returns. However, the excessive utilization of pesticides in agriculture is a severe environmental and human health problem. In the present study, effect of triazole based fungicide, propiconazole on soil physicochemical properties, stimulatory impact of propiconazole residue on soil microbial communities and soil enzyme activities (phosphatise and urease) in contaminated red sandy loam and deep black soils of paddy (Oryza sativa L.) fields at different concentrations were evaluated. The liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis showed high levels of propiconazole residues of 434 and 426 ng g-1 in red sandy loam and deep black soils, respectively. A total of 27 bacterial and 18 fungal strains were isolated from propiconazole-contaminated soils, identified by molecular markers and had their sequences deposited in the GenBank. We observed a pronounced increase in soil microbial growth, and urease and phosphatase activities at lower propiconazole concentrations up to 2 weeks post-treatment. On the contrary, the microbial growth and urease and phosphatase activities were relentlessly reduced at higher propiconazole concentrations and a long incubation period after 2 to 4 weeks of post-treatment. Overall, it was concluded that the concentration-dependency and incubation period of propiconazole would enhance the growth and activity of soil microbes and enzymes respectively and the higher application will be detrimental to the soil health.

Potential applications of plant probiotic microorganisms in agriculture and forestry.

Agriculture producers, pushed by the need for high productivity, have stimulated the intensive use of pesticides and fertilizers. Unfortunately, negative effects on water, soil, and human and animal health have appeared as a consequence of this indiscriminate practice. Plant probiotic microorganisms (PPM), also known as bioprotectants, biocontrollers, biofertilizers, or biostimulants, are beneficial microorganisms that offer a promising alternative and reduce health and environmental problems. These microorganisms are involved in either a symbiotic or free-living association with plants and act in different ways, sometimes with specific functions, to achieve satisfactory plant development. This review deals with PPM presentation and their description and function in different applications. PPM includes the plant growth promoters (PGP) group, which contain bacteria and fungi that stimulate plant growth through different mechanisms. Soil microflora mediate many biogeochemical processes. The use of plant probiotics as an alternative soil fertilization source has been the focus of several studies; their use in agriculture improves nutrient supply and conserves field management and causes no adverse effects. The species related to organic matter and pollutant biodegradation in soil and abiotic stress tolerance are then presented. As an important way to understand not only the ecological role of PPM and their interaction with plants but also the biotechnological application of these cultures to crop management, two main approaches are elucidated: the culture-dependent approach where the microorganisms contained in the plant material are isolated by culturing and are identified by a combination of phenotypic and molecular methods; and the culture-independent approach where microorganisms are detected without cultivating them, based on extraction and analyses of DNA. These methods combine to give a thorough knowledge of the microbiology of the studied environment.

Characterization of Microbial Communities in Acidified, Sulfur Containing Soils.

Over a period of three years, microbial communities in acidified soil with high sulfur content were analyzed. In soil water extracts ureolytic, proteolytic, oxidoreductive, and lipolytic activity were detected. The presented results indicate that the enzymatic activity of soil microbial communities varied considerably over time. Isolated 26 (80%) bacterial strains belonged to genus Bacillus sp. and were identified by cultivation and 16S rRNA methods. The commercially available procedures for bacterial DNA isolation from acidified soil failed, therefore a new, specific DNA isolation method was established. Ureolytic activity, detected in soil extracts as well as in isolated Bacillus sp. strains may be considered as a tool for the bioremediation of acidified soils with high sulfate content.

Effect of wood ash application on the morphological, physiological and biochemical parameters of Brassica napus L.

The present study was conducted to determine the effect of wood ash application on different parameters of Brassica napus L. including seed germination, seedling growth, fresh and dry biomass, water content in seedlings, photosynthetic pigments, soluble sugars, total protein and cell viability. In addition, the effect of wood ash on soil microflora and accumulation of trace elements in seedlings were determined. The seeds of B. napus were grown at different doses of wood ash (0, 1, 10, 25, 50 and 100 g (wood ash)/kg (soil)) and the effect on various parameters was determined. Wood ash significantly inhibited seed germination at doses above 25 g/kg and there was no germination at 100 g/kg of wood ash. At lower concentrations of wood ash, most of the growth parameters of seedlings were stimulated, but at higher concentrations of wood ash most of the studied parameters were adversely affected. Wood ash was found to be very detrimental to B. napus when applied above 25 g/kg. Wood ash application resulted in an increased bioaccumulation of trace elements in seedlings of B. napus. Almost all trace elements were significantly higher in seedlings grown in wood ash above 10 g/kg as compared to the control. An increase in total microbial count was observed with wood ash treatment which was statistically significant at 1 and 10 g/kg of wood ash. It is concluded that at very high concentration, wood ash can be detrimental to plants; however, its application at lower application rate can be recommended.

The influence of omnivorous elaterid larvae on the microbial carbon cycle in different forest soils.

Data are presented on the influence of Athous subfuscus larvae (Coleoptera, Elateridae) on the microbial carbon cycle in the biotically most active horizons of three contrasting beech forest soils: the Ah horizon of a mull soil on limestone (Göttinger Wald, FRG), the F/H horizon of a moder soil on new red sandstone (Solling area, FRG) and in the F/H horizon of a lime ameliorated area close to the second site. Gut content analyses demonstrated that the larvae of A. subfuscus are humiphagous and that this unspecific feeding behaviour is widely independent of soil conditions. Differences in 14C incorporation demonstrated that only the larvae in the F/H horizon of the limed moder soil directly affected primary decomposer organisms. However, the burrowing activity of the larvae in the topsoil indirectly modified the time course of beech leaf-litter decomposition in the litter layer of all three soils. The microflora of the mull soil contained 2.6%, that of the moder soil 0.7% and that of the limed moder soil 2.2% of total C. The metabolic quotient (qCO2, 10°C) of the soil microflora was 0.0010 (mgCO2-C·mg-1 biomass-C·h-1) in the mull soil, 0.0034 in the moder soil and 0.0012 in the limed moder soil. The A. subfuscus larvae generally reduced the size of the microbial C pool (<-30%) and increased the metabolic quotient of the microflora (>+50%). Considering these soil-independent effects of A. subfuscus on the C turnover of the soil microflora, the burrowing activity of humiphagous soil arthropods may generally increase nutrient availability to primary producers. The results of this study reveal that some of the micro- and mesoscale effects of humiphagous arthropods on the microbial carbon turnover in beech forest soils are surprisingly similar, even under very different soil conditions. The long-term modification of the time course of leaf litter decomposition, in contrast, indicates that the influence of humiphagous arthropods on the formation of the humus layer is soil-specific. There are profound differences in the role of humiphagous arthropods in limed moder soils and in naturally base-rich soils. It is concluded that liming increases competition within the microfloral population due to accelerated humification. The negative effect of A. subfuscus on 14C mineralization in the limed substrate could thus be explained by its effects on a microflora that was strongly limited by the availability of carbon.

The Role of Secondary Invaders in Meloidogyne incognita Infection.

Secondary microbial invasion of tomato plants inoculated with 6,000 Meloidogyne incognita larvae caused 75 and 48% weight reduction of foliage and roots, respectively. Under aseptic conditions the same number of larvae caused only 37% reduction of foliage and increased root weight by 50%.