Efficacy of Azadirachtin in the Integrated Management of the Root Knot Nematode Meloidogyne incognita on Short- and Long-Cycle Crops.

Activity of azadirachtin on phytoparasitic nematodes has been documented for some decades, but the relationship between its nematicidal efficacy and crop cycle length has not yet been clarified. This study aimed to assess the efficacy of an azadirachtin-based nematicide, for controlling the infestation of the root-knot nematode Meloidogyne incognita, on the short- and long-cycle crops, lettuce and tomato, respectively. Experiments on lettuce and tomato were carried out in a greenhouse infested by M. incognita, including non-treated soil, or treated with the nematicide fluopyram, as controls. In the experiment on the short-cycle lettuce crop, the azadirachtin product effectively suppressed M. incognita infestation and increased crop yield, without significant differences from fluopyram. In the tomato crop, both azadirachtin and fluopyram were not able to control nematode infestation, but resulted in significantly higher yields. Data from this study indicated that azadirachtin can be a valid alternative to fluopyram and other nematicides, for root-knot nematode control in short-cycle crops. Integration of azadirachtin with a synthetic nematicide or nematode-suppressive agronomical techniques, should be more suitable to long-cycle crops.

Synergistic Effects of Trichoderma harzianum, 1,3 Dichloropropene and Organic Matter in Controlling the Root-Knot Nematode Meloidogyne incognita on Tomato.

Environmental concerns raised by synthetic nematicides are encouraging integrated management strategies based on their combination with non-chemical control tools, such as biocontrol agents and/or organic amendments. In this study, the combination of the fumigant 1,3-dichloropropene (1,3-D) with a commercial formulation of the biocontrol agent Trichoderma harzianum (TH) and an organic fertilizer (OF) was investigated in two consecutive tomato crops for its effect on the root-knot nematode Meloidogyne incognita and plant growth and yield. The application of 1,3-D was only performed on the first crop, while TH and OF were provided to both crops. Almost all treatments significantly reduced nematode infestation in both crops, though the greatest nematicidal effect was caused by a combination of the three products. The treatment with 1,3-D limited its nematicidal efficacy to the first crop only. Fumigant integration with TH and OF also resulted in the greatest increases of plant growth and yield. Therefore, the integrated management of root-knot nematodes with a soil fumigant, a bionematicide as T. harzianum and a source of organic matter demonstrated effective nematode suppression though limiting the number of chemical applications.

Impact of Super-High Density Olive Orchard Management System on Soil Free-Living and Plant-Parasitic Nematodes in Central and South Italy.

The soil nematode community plays an important role in ecosystem services. The objective of this study was to assess the effect of Super-high density (SHD) olive orchards on the nematode community in five sites with different soils, climates, and cultivars. At each site, the SHD management system was compared to the adjacent olive orchard traditional (TRAD) system, in which the same soil management and phytosanitary measures were applied. Soil management was assessed by total organic carbon content (TOC), while the soil nematode community was evaluated using the nematode taxa abundances and soil nematode indicators. TOC was significantly decreased in the SHD olive orchard system compared to TRAD in the sites characterized by conventional tillage and mineral fertilization. The two-way ANOSIM analysis on nematode abundance showed no difference between the two olive management methods, instead showing only a significant difference per site mainly due to variabilities in plant-parasitic nematode assemblage. However, a negative impact of SHD management was evident in environments stressed by summer droughts and conventional tillage: the ratio of obligate plant-parasites to bacterivores and fungivores (Pp/(B+F)) was significantly higher in SHD than in the TRAD olive orchard system, and the prey-to-predator θ mass ratio showed the lowest values in the sites under organic fertilization or green manure. The canonical correspondence analysis showed that the free-living nematodes were only slightly affected by SHD olive orchards; instead, the presence of plant-parasitic nematodes families such as Telotylenchidae, Paratylenchidae, Meloidogynidae, and Criconematidae was favored, in comparison to Longidoridae, Heteroderidae, and Pratylenchidae.

Metabolites of a Drechslera sp. endophyte with potential as biocontrol and bioremediation agent.

Endophytic fungi have several well-established beneficial effects on plant health and growth, and are a huge source of bioactive compounds. The endophyte Drechslera sp. strain 678, isolated from the roots of an Australian native grass Neurachne alopecuroidea, demonstrated efficacy against four plant pathogens (Pythium ultimum, Rhizoctonia solani, Botrytis cinerea, Alternaria alternata). In addition, strain 678 was capable of degrading a common additive used in gasoline, known as methyl tertiary-butyl ether (MtBE). Thus, the organic extracts obtained from the culture filtrate of strain 678 were studied. Metabolomic analysis revealed the presence of two major bioactive metabolites, monocerin and an alkynyl substituted epoxycyclohexenone derivative, which showed good antifungal activity. The Drechslera sp. strain 678 and its compounds show promise for applications in biocontrol and bioremediation activities in agriculture or as a remediation option for MtBE contamination in soil.

Biochar-derived smoke-water exerts biological effects on nematodes, insects, and higher plants but not fungi.

The pyrolysis of organic feedstock yields the solid fraction biochar, bio-oils, and a volatile fraction that can be reused for energetic purposes or technological applications in agro-ecosystems in the form of smoke-water (SW). In this study, 10 SW types were created from five organic feedstocks (i.e. cellulose, wood sawdust, olive mill residues, maize, and alfalfa litter) at two pyrolysis temperatures (i.e. 300 and 500 °C). We characterized SW using liquid chromatography (LC)-electrospray ionization-time-of-flight (TOF) mass spectrometry (MS) combined with a multi-species bioassay including five crop plants, four fungi, one root-knot nematode (Meloidogyne incognita), and the olive fly pest (Bactrocera oleae). All SW types were acidic, exhibiting a pH range of 1.9-4.6. LC-MS analysis revealed differences in the chemical profiles of SW types in relation to the organic feedstock type and pyrolysis temperature. All SW types exerted concentration-dependent effects on crops, with evident phytotoxic activity at high concentrations. Conversely, they exerted stimulatory effects when diluted with water at ratios ranging from 1:100 to 1:1000. Moreover, all SW types displayed slight or null fungitoxic activity. On the contrary, SW strongly inhibited egg hatching by M. incognita after 72 and 144 h of incubation. The strongest inhibition was found for olive mill SW, and the weakest effect was noted for alfalfa SW. Finally, the application of SW over fresh olives reversed the attraction of B. oleae adults, demonstrating a strong repellent effect toward this pest. Nevertheless, only olive mill SW consistently attracted B. oleae. In conclusion, biochar SW exhibited notable biological activities and potential applications for plant growth promotion, if opportunely diluted, and for the control of root-knot nematodes and olive fruit flies.

The Application of Trichoderma Strains or Metabolites Alters the Olive Leaf Metabolome and the Expression of Defense-Related Genes.

Biocontrol fungal strains of the genus Trichoderma can antagonize numerous plant pathogens and promote plant growth using different mechanisms of action, including the production of secondary metabolites (SMs). In this work we analyzed the effects of repeated applications of selected Trichoderma strains or SMs on young olive trees on the stimulation of plant growth and on the development of olive leaf spot disease caused by Fusicladium oleagineum. In addition, metabolomic analyses and gene expression profiles of olive leaves were carried out by LC-MS Q-TOF and real-time RT-PCR, respectively. A total of 104 phenolic compounds were detected from olive leave extracts and 20 were putatively identified. Targeted and untargeted approaches revealed significant differences in both the number and type of phenolic compounds accumulated in olive leaves after Trichoderma applications, as compared to water-treated plants. Different secoiridoids were less abundant in treated plants than in controls, while the accumulation of flavonoids (including luteolin and apigenin derivatives) increased following the application of specific Trichoderma strain. The induction of defense-related genes, and of genes involved in the synthesis of the secoiridoid oleuropein, was also analyzed and revealed a significant variation of gene expression according to the strain or metabolite applied.

Effect of Trichoderma Bioactive Metabolite Treatments on the Production, Quality, and Protein Profile of Strawberry Fruits.

Fungi of the genus Trichoderma produce secondary metabolites having several biological activities that affect plant metabolism. We examined the effect of three Trichoderma bioactive metabolites (BAMs), namely, 6-pentyl-α-pyrone (6PP), harzianic acid (HA), and hydrophobin 1 (HYTLO1), on yield, fruit quality, and protein representation of strawberry plants. In particular, 6PP and HA increased the plant yield and number of fruits, when compared to control, while HYTLO1 promoted the growth of the roots and increased the total soluble solids content up to 19% and the accumulation of ascorbic acid and cyanidin 3-O-glucoside in red ripened fruits. Proteomic analysis showed that BAMs influenced the representation of proteins associated with the protein metabolism, response to stress/external stimuli, vesicle trafficking, carbon/energy, and secondary metabolism. Results suggest that the application of Trichoderma BAMs affects strawberry plant productivity and fruit quality and integrate previous observations on deregulated molecular processes in roots and leaves of Trichoderma-treated plants with original data on fruits.

Milk Metabolomics Reveals Potential Biomarkers for Early Prediction of Pregnancy in Buffaloes Having Undergone Artificial Insemination.

This study aimed to identify potential biomarkers for early pregnancy diagnosis in buffaloes subjected to artificial insemination (AI). The study was carried out on 10 pregnant and 10 non-pregnant buffaloes that were synchronized by Ovsynch-Timed Artificial Insemination Program and have undergone the first AI. Furthermore, milk samples were individually collected ten days before AI (the start of the synchronization treatment), on the day of AI, day 7 and 18 after AI, and were analyzed by LC-MS. Statistical analysis was carried out by using Mass Profile Professional (Agilent Technologies, Santa Clara, CA, USA). Metabolomic analysis revealed the presence of several metabolites differentially expressed between pregnant and non-pregnant buffaloes. Among these, a total of five metabolites were identified by comparison with an online database and a standard compound as acetylcarnitine (3-Acetoxy-4-(trimethylammonio)butanoate), arginine-succinic acid hydrate, 5'-O-{[3-({4-[(3aminopropyl)amino]butyl}amino)propyl]carbamoyl}-2'-deoxyadenosine, N-(1-Hydroxy-2-hexadecanyl)pentadecanamide, and N-[2,3-Bis(dodecyloxy)propyl]-L-lysinamide). Interestingly, acetylcarnitine was dominant in milk samples collected from non-pregnant buffaloes. The results obtained from milk metabolic profile and hierarchical clustering analysis revealed significant differences between pregnant and non-pregnant buffaloes, as well as in the metabolite expression. Overall, the findings indicate the potential of milk metabolomics as a powerful tool to identify biomarkers of early pregnancy in buffalo undergoing AI.

Antibiofilm Activity of a Trichoderma Metabolite against Xanthomonas campestris pv. campestris, Alone and in Association with a Phage.

Biofilm protects bacteria against the host's immune system and adverse environmental conditions. Several studies highlight the efficacy of lytic phages in the prevention and eradication of bacterial biofilms. In this study, the lytic activity of Xccφ1 (Xanthomonas campestris pv. campestris-specific phage) was evaluated in combination with 6-pentyl-α-pyrone (a secondary metabolite produced by Trichoderma atroviride P1) and the mineral hydroxyapatite. Then, the antibiofilm activity of this interaction, called a φHA6PP complex, was investigated using confocal laser microscopy under static and dynamic conditions. Additionally, the mechanism used by the complex to modulate the genes (rpf, gumB, clp and manA) involved in the biofilm formation and stability was also studied. Our results demonstrated that Xccφ1, alone or in combination with 6PP and HA, interfered with the gene pathways involved in the formation of biofilm. This approach can be used as a model for other biofilm-producing bacteria.

Methyl t-butyl ether-degrading bacteria for bioremediation and biocontrol purposes.

A total of fifteen potential methyl t-butyl ether (MtBE)-degrading bacterial strains were isolated from contaminated soil. They have been identified as belonging to the genera Bacillus, Pseudomonas, Kocuria, Janibacter, Starkeya, Bosea, Mycolicibacterium, and Rhodovarius. Bacillus aryabhattai R1B, S. novella R8b, and M. mucogenicum R8i were able to grow using MtBE as carbon source, exhibiting different growth behavior and contaminant degradation ability. Their biocontrol ability was tested against various fungal pathogens. Both S. novella R8b and B. aryabhattai were effective in reducing the development of necrotic areas on leaves within 48 hours from Botritys cinerea and Alternaria alternata inoculation. Whereas, M. mucogenicum effectively controlled B. cinerea after 72 hours. Similar results were achieved using Pythium ultimum, in which the application of isolated bacteria increased seed germination. Only M. mucogenicum elicited tomato plants resistance against B. cinerea. This is the first report describing the occurrence of bioremediation and biocontrol activities in M. mucogenicum, B. aryabhattai and S. novella species. The production of maculosin and its antibiotic activity against Rhizoctonia solani has been reported for first time from S. novella. Our results highlight the importance of multidisciplinary approaches to achieve a consistent selection of bacterial strains useful for plant protection and bioremediation purposes.

Application of Trichoderma Strains and Metabolites Enhances Soybean Productivity and Nutrient Content.

Trichoderma fungi are effectively marketed worldwide as biocontrol agents and plant biostimulants on numerous crops due to their demonstrated effects in direct antagonism against fungal pathogens and plant growth promotion. Here, we examined the effects of single and combined applications of Trichoderma strains and their bioactive metabolites (BAMs) harzianic acid (HA), 6-pentyl-α-pyrone (6PP), and hydrophobin1 (HYTLO1) on the growth, yield, and nutrient uptake of soybean plants. Significant promotion of plant growth (up to 39%), as well as an increase in mineral content, was achieved with BAMs, used alone or combined with T. harzianum. Interestingly, the treatments also increased the level of fatty acids (oleic, linolenic, 11-eicosenoic, and stearic). This work demonstrates the usefulness of natural compound and microbe combinations to enhance oilseed productivity, and reports for the first time the ability of Trichoderma and/or its BAMs to increase the lipid content in harvested seeds.

Biochars from olive mill waste have contrasting effects on plants, fungi and phytoparasitic nematodes.

Olive mill waste (OMW), a byproduct from the extraction of olive oil, causes serious environmental problems for its disposal, and extensive efforts have been made to find cost-effective solutions for its management. Biochars produced from OMW were applied as soil amendment and found in many cases to successfully increase plant productivity and suppress diseases. This work aims to characterize biochars obtained by pyrolysis of OMW at 300 °C to 1000 °C using 13C NMR spectroscopy, LC-ESI-Q-TOF-MS and SEM (Scanning Electron Microscopy). Chemical characterization revealed that biochar composition varied according to the increase of pyrolysis temperature (PT). Thermal treated materials showed a progressive reduction of alkyl C fractions coupled to the enrichment in aromatic C products. In addition, numerous compounds present in the organic feedstock (fatty acids, phenolic compounds, triterpene acids) reduced (PT = 300 °C) or completely disappeared (PT ≥ 500 °C) in biochars as compared to untreated OMW. PT also affected surface morphology of biochars by increasing porosity and heterogeneity of pore size. The effects of biochars extracts on the growth of different organisms (two plants, one nematode and four fungal species) were also evaluated. When tested on different living organisms, biochars and OMW showed opposite effects. The root growth of Lepidium sativum and Brassica rapa, as well as the survival of the nematode Meloidogyne incognita, were inhibited by the untreated material or biochar produced at 300 °C, but toxicity decreased at higher PTs. Conversely, growth of Aspergillus, Fusarium, Rhizoctonia and Trichoderma fungi was stimulated by organic feedstock, while being inhibited by thermally treated biochars. Our findings showed a pattern of association between specific biochar chemical traits and its biological effects that, once mechanistically explained and tested in field conditions, may lead to effective applications in agriculture.

Root Exudates of Stressed Plants Stimulate and Attract Trichoderma Soil Fungi.

Plant roots release complex mixtures of bioactive molecules, including compounds that affect the activity and modify the composition of the rhizosphere microbiome. In this work, we investigated the initial phase of the interaction between tomato and an effective biocontrol strain of Trichoderma harzianum (T22). We found that root exudates (RE), obtained from plants grown in a split-root system and exposed to various biotic and abiotic stress factors (wounding, salt, pathogen attack), were able to stimulate the growth and act as chemoattractants of the biocontrol fungus. On the other hand, some of the treatments did not result in an enhanced chemotropism on Fusarium oxysporum f. sp. lycopersici, indicating a mechanism that may be selective for nonpathogenic microbes. The involvement of peroxidases and oxylipins, both known to be released by roots in response to stress, was demonstrated by using RE fractions containing these molecules or their commercial purified analogs, testing the effect of an inhibitor, and characterizing the complex pattern of these metabolites released by tomato roots both locally and systemically.

How ecosystems change following invasion by Robinia pseudoacacia: Insights from soil chemical properties and soil microbial, nematode, microarthropod and plant communities.

Biological invasions are a global threat to biodiversity. Since the spread of invasive alien plants may have many impacts, an integrated approach, assessing effects across various ecosystem components, is needed for a correct understanding of the invasion process and its consequences. The nitrogen-fixing tree Robinia pseudoacacia (black locust) is a major invasive species worldwide and is used in forestry production. While its effects on plant communities and soils are well known, there have been few studies on soil fauna and microbes. We investigated the impacts of the tree on several ecosystem components, using a multi-trophic approach to combine evidence of soil chemical properties and soil microbial, nematode, microarthropod and plant communities. We sampled soil and vegetation in managed forests, comparing those dominated by black locust with native deciduous oak stands. We found qualitative and quantitative changes in all components analysed, such as the well-known soil nitrification and acidification in stands invaded by black locust. Bacterial richness was the only component favoured by the invasion. On the contrary, abundance and richness of microarthropods, richness of nematodes, and richness and diversity of plant communities decreased significantly in invaded stands. The invasion process caused a compositional shift in all studied biotic communities and in relationships between the different ecosystem components. We obtained clear insights into the effects of invasion of managed native forests by black locust. Our data confirms that the alien species transforms several ecosystem components, modifying the plant-soil community and affecting biodiversity at different levels. Correct management of this aggressive invader in temperate forests is urgently required.