Reduction In Fungal Biomass Research Articles

An RNA interference (RNAi) target with potential to control Dothistroma needle blight

Spray-induced gene silencing (SIGS) involves exogenous spray applications of double-stranded RNA (dsRNA) molecules targeting specific genes, such as those essential to pathogens. This technique has shown great potential for controlling fungal pathogens of horticultural and agricultural plants, but few studies have evaluated its application to forest pathogens. Here we report the first demonstration of exogenous application of dsRNA molecules targeting a gene required for virulence in the foliar pine pathogen Dothistroma septosporum, the causal agent of Dothistroma needle blight (DNB). Constructs expressing sense and antisense dsRNAs were generated targeting a 509 bp fragment of the dothistromin toxin regulatory gene (DsAflR) in D. septosporum. DsAflR is required for the production of dothistromin, a phytotoxin essential for full pathogen virulence. We found that exogenous dsRNA is taken up and the RNA silencing mechanism is functional in D. septosporum through a series of in vitro and in planta trials, such as monitoring the uptake of fluorescently labelled DsAflR-dsRNA into the cell by confocal microscopy and quantifying the extent of gene silencing by quantitative Polymerase Chain Reaction (qPCR). In vitro applications of DsAflR-dsRNA to D. septosporum reduced the accumulation of DsAflR mRNA compared to controls and, in some cases, were found to be statistically significant, despite a high level of variability in the results. Our study also demonstrated a reduction in DNB symptoms on infected pine needles sprayed with DsAflR-dsRNA, which was further supported by a significant reduction in fungal biomass. This preliminary study highlights the potential for SIGS as a future management scheme for controlling the forest pathogen D. septosporum, as well as other forest pathogens.

Enhancing plant defense using rhizobacteria in processing tomatoes: a bioprospecting approach to overcoming Early Blight and Alternaria toxins.

Plant growth-promoting rhizobacteria (PGPR) with antagonistic activity toward plant pathogenic fungi are valuable candidates for the development of novel plant protection products based on biocontrol activity. The very first step in the formulation of such products is to screen the potential effectiveness of the selected microorganism(s). In this study, non-pathogenic rhizobacteria were isolated from the rhizosphere of tomato plants and evaluated for their biocontrol activity against three species of mycotoxin-producing Alternaria. The assessment of their biocontrol potential involved investigating both fungal biomass and Alternaria toxin reduction. A ranking system developed allowed for the identification of the 12 best-performing strains among the initial 85 isolates. Several rhizobacteria showed a significant reduction in fungal biomass (up to 76%) and/or mycotoxin production (up to 99.7%). Moreover, the same isolates also demonstrated plant growth-promoting (PGP) traits such as siderophore or IAA production, inorganic phosphate solubilization, and nitrogen fixation, confirming the multifaceted properties of PGPRs. Bacillus species, particularly B. amyloliquefaciens and two strains of B. subtilis, showed the highest efficacy in reducing fungal biomass and were also effective in lowering mycotoxin production. Isolates such as Enterobacter ludwigii, Enterobacter asburiae, Serratia nematodiphila, Pantoea agglomerans, and Kosakonia cowanii showed moderate efficacy. Results suggest that by leveraging the diverse capabilities of different microbial strains, a consortium-based approach would provide a broader spectrum of effectiveness, thereby signaling a more encouraging resolution for sustainable agriculture and addressing the multifaceted nature of crop-related biotic challenges.

The northern corn leaf blight resistance gene Ht1 encodes an nucleotide-binding, leucine-rich repeat immune receptor.

Northern corn leaf blight, caused by the fungal pathogen Exserohilum turcicum, is a major disease of maize. The first major locus conferring resistance to E.turcicum race 0, Ht1, was identified over 50 years ago, but the underlying gene has remained unknown. We employed a map-based cloning strategy to identify the Ht1 causal gene, which was found to be a coiled-coil nucleotide-binding, leucine-rich repeat (NLR) gene, which we named PH4GP-Ht1. Transgenic testing confirmed that introducing the native PH4GP-Ht1 sequence to a susceptible maize variety resulted in resistance to E.turcicum race 0. A survey of the maize nested association mapping genomes revealed that susceptible Ht1 alleles had very low to no expression of the gene. Overexpression of the susceptible B73 allele, however, did not result in resistant plants, indicating that sequence variations may underlie the difference between resistant and susceptible phenotypes. Modelling of the PH4GP-Ht1 protein indicated that it has structural homology to the Arabidopsis NLR resistance gene ZAR1, and probably forms a similar homopentamer structure following activation. RNA sequencing data from an infection time course revealed that 1 week after inoculation there was a threefold reduction in fungal biomass in the PH4GP-Ht1 transgenic plants compared to wild-type plants. Furthermore, PH4GP-Ht1 transgenics had significantly more inoculation-responsive differentially expressed genes than wild-type plants, with enrichment seen in genes associated with both defence and photosynthesis. These results demonstrate that the NLR PH4GP-Ht1 is the causal gene underlying Ht1, which represents a different mode of action compared to the previously reported wall-associated kinase northern corn leaf blight resistance gene Htn1/Ht2/Ht3.

Computational Strategy for Minimizing Mycotoxins in Cereal Crops: Assessment of the Biological Activity of Compounds Resulting from Virtual Screening.

Cereal crops are frequently affected by toxigenic Fusarium species, among which the most common and worrying in Europe are Fusarium graminearum and Fusarium culmorum. These species are the causal agents of grain contamination with type B trichothecene (TCTB) mycotoxins. To help reduce the use of synthetic fungicides while guaranteeing low mycotoxin levels, there is an urgent need to develop new, efficient and environmentally-friendly plant protection solutions. Previously, F. graminearum proteins that could serve as putative targets to block the fungal spread and toxin production were identified and a virtual screening undertaken. Here, two selected compounds, M1 and M2, predicted, respectively, as the top compounds acting on the trichodiene synthase, a key enzyme of TCTB biosynthesis, and the 24-sterol-C-methyltransferase, a protein involved in ergosterol biosynthesis, were submitted for biological tests. Corroborating in silico predictions, M1 was shown to significantly inhibit TCTB yield by a panel of strains. Results were less obvious with M2 that induced only a slight reduction in fungal biomass. To go further, seven M1 analogs were assessed, which allowed evidencing of the physicochemical properties crucial for the anti-mycotoxin activity. Altogether, our results provide the first evidence of the promising potential of computational approaches to discover new anti-mycotoxin solutions

Long-term cattle grazing shifts the ecological state of forest soils.

Cattle grazing profoundly affects abiotic and biotic characteristics of ecosystems. While most research has been performed on grasslands, the effect of large managed ungulates on forest ecosystems has largely been neglected. Compared to a baseline seminatural state, we investigated how long‐term cattle grazing of birch forest patches affected the abiotic state and the ecological community (microbes and invertebrates) of the soil subsystem. Grazing strongly modified the soil abiotic environment by increasing phosphorus content, pH, and bulk density, while reducing the C:N ratio. The reduced C:N ratio was strongly associated with a lower microbial biomass, mainly caused by a reduction of fungal biomass. This was linked to a decrease in fungivorous nematode abundance and the nematode channel index, indicating a relative uplift in the importance of the bacterial energy‐channel in the nematode assemblages. Cattle grazing highly modified invertebrate community composition producing distinct assemblages from the seminatural situation. Richness and abundance of microarthropods was consistently reduced by grazing (excepting collembolan richness) and grazing‐associated changes in soil pH, Olsen P, and reduced soil pore volume (bulk density) limiting niche space and refuge from physical disturbance. Anecic earthworm species predominated in grazed patches, but were absent from ungrazed forest, and may benefit from manure inputs, while their deep vertical burrowing behavior protects them from physical disturbance. Perturbation of birch forest habitat by long‐term ungulate grazing profoundly modified soil biodiversity, either directly through increased physical disturbance and manure input or indirectly by modifying soil abiotic conditions. Comparative analyses revealed the ecosystem engineering potential of large ungulate grazers in forest systems through major shifts in the composition and structure of microbial and invertebrate assemblages, including the potential for reduced energy flow through the fungal decomposition pathway. The precise consequences for species trophic interactions and biodiversity–ecosystem function relationships remain to be established, however.

A Novel In Planta Enrichment Method Employing Fusarium graminearum-Infected Wheat Spikes to Select for Competitive Biocontrol Bacteria.

This work introduces an alternative workflow for the discovery of novel bacterial biocontrol agents in wheat against Fusarium head blight (FHB). Unlike the mass testing of isolate collections, we started from a diverse inoculum by extracting microbiomes from ears of field-grown plants at grain filling stage. Four distinct microbial communities were generated which were exposed to 3 14-day culture-independent experimental enrichments on detached wheat spikes infected with F. graminearum PH1. We found that one bacterial community reduced infection symptoms after 3 cycles, which was chosen to subsequently isolate bacteria through limiting dilution. All 94 isolates were tested in an in vitro and in planta assay, and a selection of 14 isolates was further tested on detached ears. The results seem to indicate that our enrichment approach resulted in bacteria with different modes-of-action in regard to FHB control. Erwinia persicina isolate C3 showed a significant reduction in disease severity (Fv/Fm), and Erwinia persicina C3 and Pseudomonas sp. B3 showed a significant reduction in fungal biomass (cGFP). However, the mycotoxin analysis of both these treatments showed no reduction in DON levels. Nevertheless, Pantoea ananatis H3 and H11 and Erwinia persicina H2 were able to reduce DON concentrations by more than 50%, although these effects were not statistically significant. Lastly, Erwinia persicina H2 also showed a significantly greater glucosylation of DON to the less phytotoxic DON-3G. The bacterial genera isolated through the enrichment cycles have been reported to dominate microbial communities that develop in open habitats, showing strong indications that the isolated bacteria can reduce the infection pressure of F. graminearum on the spike phyllosphere.

Antagonistic and growth enhancement activities of native Pseudomonas spp. against soil and tuber-borne diseases of potato (Solanum tuberosum L.)

BackgroundSoil and tuber-borne diseases are serious threat for potato cultivation worldwide which causes severe economical losses in terms of yield, quantity and quality. Generally, these diseases managed by chemical pesticide, are a major concern for human health and environment. Therefore, finding an eco-friendly alternative management strategies are necessary. The native bacterial isolates collected from different crops rhizosphere soil were evaluated against Rhizoctonia solani, Sclerotinia sclerotiorum, Sclerotium rolfsii and Fusarium spp and were also tested for their growth enhancement attributes on potato crop and phosphate solubilising efficiency.ResultsBased on the morphological and phenotypic characters, most of the isolates were identified as Pseudomonas spp (18 isolates). A total of twenty-two bacterial isolates were screened for bio-control activity in dual culture assay. Isolate Pf14 showed the highest mycelial inhibitory potential (ranged from 62.2 to 59.3%) against R. solani, S. sclerotiorum, S. rolfsii and Fusarium spp. In sealed plate assay, Pf14 produced antifungal volatile compounds that significantly inhibited mycelial growth (ranged from > 80 to > 50%). Maximum reduction in fungal biomass (ranged from > 80%) was observed in King’s broth in shake liquid culture in all the pathogens. Cell-free culture filtrate of the selected isolate inhibited mycelial growth ranged from 68.9 to 42.6% of the tested pathogens with 48-h old culture filtrate. Additionally, the isolates exhibited higher phosphorus solubilizing efficiency on PVK media. Under field conditions, talc based formulation of Pf14 showed enhanced agronomical characters and inhibits black scurf severity up to 67.59%. This treatment also recorded a highest tuber yield (21.90 t/ha) with increase of 15.38% in comparison to untreated control.ConclusionOverall, antagonistic bacterium Pf14 can be recommended as bio fertilizers for eco-friendly management of major potato fungal diseases as well as increasing marketable yield and used as an alternative to the pesticides and chemical fertilizers.

English

Macrophomina phaseolina (Tassi) Goid. is a soil-borne fungal pathogen causing diseases in more than 500 plant species. The present study aimed to identify possible antifungal constituents in different parts of billygoat-weed (Ageratum conyzoides L.) through bioassays guided fractionation for the control of M. phaseolina. Extracts of different parts of the weed were made in methanol and antifungal bioassays were conducted using 1, 2, 3, 4 and 5% concentrations of the extract. Stem extract caused the highest inhibition in fungal biomass (20–83%) followed by leaf extract (16–67%). Methanolic stem extract was partitioned using four organic solvents namely n-hexane, chloroform, ethyl acetate and n-butanol. Bioassays carried out with different concentrations (3.125, 6.25, 12.5, 25, 50, 100 and 200 mg mL-1) of the sub-fractions of stem extract revealed the highest antifungal potential of chloroform sub-fraction with 56–93% reduction in the fungal biomass followed by n-butanol, ethyl acetate and n-hexane sub-fractions causing 24–76%, 7–75% and 5–70% reduction in fungal biomass over control, respectively. Chloroform sub-fraction with the highest antifungal potential was analyzed by GC-MS. Out of 10 compounds identified in this sub-fraction, 2H-1-benzopyran, 7-dimethoxy-2,2-dimethyl- (27.58%) was the most abundant followed by hexadecanoic acid, methyl ester (18.85%); 11-octadecenoic acid, methyl ester (15.28%) and 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester (10.88%), which could be responsible for antifungal activity. © 2021 Friends Science Publishers

ANTIFUNGAL ACTIVITY OF LEAF EXTRACT OF Cannabis sativa AGAINST Aspergillus flavipes

Cannabis sativa L. is a medicinally important weed of family Cannabaceae generally grows along road-sides and waste-lends in Punjab, Pakistan. In the present study, antifungal effect of leaf extract of this weed was assessed against Aspergillus flavipes. Methanolic leaf extract of the weed was partitioned into five fractions using organic solvents of variable polarities. A range of concentrations (1.562 to 200 mg mL-1) of each fraction was used in laboratory bioassays. n-Butanol fraction showed the highest antifungal activity followed by chloroform and n-hexane fractions causing 68–82%, 52–82% and 42–82% decrease in biomass of A. flavipes. Ethyl acetate showed a moderate antifungal potential while aqueous fraction showed the least antifungal activity causing 47–76% and 38–73% reduction in fungal biomass, respectively. This study concludes that n-butanol fraction of leaf extract of C. sativa is highly effective in controlling growth of A. flavipes.

MLO Differentially Regulates Barley Root Colonization by Beneficial Endophytic and Mycorrhizal Fungi.

Loss-of-function alleles of MLO (Mildew Resistance Locus O) confer broad-spectrum resistance to foliar infections by powdery mildew pathogens. Like pathogens, microbes that establish mutually beneficial relationships with their plant hosts, trigger the induction of some defense responses. Initially, barley colonization by the root endophyte Serendipita indica (syn. Piriformospora indica) is associated with enhanced defense gene expression and the formation of papillae at sites of hyphal penetration attempts. This phenotype is reminiscent of mlo-conditioned immunity in barley leaf tissue and raises the question whether MLO plays a regulatory role in the establishment of beneficial interactions. Here we show that S. indica colonization was significantly reduced in plants carrying mlo mutations compared to wild type controls. The reduction in fungal biomass was associated with the enhanced formation of papillae. Moreover, epidermal cells of S. indica-treated mlo plants displayed an early accumulation of iron in the epidermal layer suggesting increased basal defense activation in the barley mutant background. Correspondingly, the induction of host cell death during later colonization stages was impaired in mlo colonized plants, highlighting the importance of the early biotrophic growth phase for S. indica root colonization. In contrast, the arbuscular mycorrhizal fungus Funneliformis mosseae displayed a similar colonization morphology on mutant and wild type plants. However, the frequency of mycorrhization and number of arbuscules was higher in mlo-5 mutants. These findings suggest that MLO differentially regulates root colonization by endophytic and AM fungi.

Glyphosate but not Roundup® harms earthworms (Eisenia fetida)

Glyphosate is the active ingredient in Roundup® formulations. While multiple studies have documented the toxicity, environmental persistence, and tendency to spread for glyphosate and Roundup®, few studies have compared the toxicity of glyphosate-based formulations to the toxicity of pure glyphosate for soil invertebrates, which contact both the herbicide and the formulations. Hundreds of formulations exist; their inert ingredients are confidential; and glyphosate persists in our food, water, and soil. In this experiment, we held glyphosate type and concentration constant, varying only formulation. Using Roundup Ready-to-Use III®, Roundup Super Concentrate®, and pure glyphosate, we delivered 26.3 mg glyphosate in the form of isopropylamine salt per kg of soil to compost worms (Eisenia fetida). We found that worms living in soil spiked with pure glyphosate lost 14.8–25.9% of their biomass and survived a stress test for 22.2–33.3% less time than worms living in uncontaminated soil. Worms living in soil spiked with Roundup Ready-to-Use III® and Roundup Super Concentrate® did not lose body mass and survived the stress test as well as worms living in uncontaminated soil. No contaminant affected soil microbial or fungal biomass over the 40-day period of this experiment. We suggest that the nitrates and phosphates in the formulations offset the toxic effects of glyphosate by spurring microbial growth and speeding glyphosate degradation. We also found a 26.5–41.3% reduction in fungal biomass across all treatments over the course of this experiment, suggesting that the worms consumed fungi and spores.

Anti-mycotic potential of Trichoderma spp. and leaf biomass of Azadirachta indica against the charcoal rot pathogen, Macrophomina phaseolina (Tassi) Goid in cowpea

Macrophomina phaseolina (Tassi) Goid is a destructive pathogen of cowpea that causes serious charcoal rot disease with significant yield losses. Antifungal activity of three indigenous Ascomycetes viz., Trichoderma harzianum, T. viride, and T. hamatum, and two Meliaceae members, i.e., Melia azedarach L. and Azadirachta indica L. were assessed against the pathogen. Laboratory screening trials with cell-free culture filtrate showed the maximum reduction in growth of M. phaseolina with T. harzianum, followed by T. viride. Various concentrations (1–5%) of methanolic leaf extract of A. indica showed more reduction in fungal biomass than M. azedarach. Pot experiment was performed by T. harzianum, T. viride, and dry leaf biomass of A. indica against M. phaseolina. Results revealed that potted soil amended with T. harzianum in combination with 1–3% dry leaf biomass of A. indica held a significant potential to decrease disease incidence to 20–25% and improve plant growth attributes up to fourfolds over positive control inoculated with M. phaseolina only. Physiology of the host plant was altered due to the incorporation of various soil amendments resulting in reduced activities of antioxidant enzymes (catalase, peroxidase, polyphenol oxidase, and phenylalanine ammonia lyase). It was concluded that fungal antagonists and allelopathic chemicals would be an effective and eco-friendly means of managing the charcoal rot disease.

ANTIFUNGAL POTENTIAL OF A BRASSICACEOUS WEED Sisymbrium irio AGAINST Macrophomina phaseolina

ABSTRACT Macrophomina phaseolina, a soil-borne plant pathogen, has the ability to cause diseases in about 500 plant species. Unfortunately, so far no registered fungicide is available against this fungal pathogen. In the present study, different extracts of Sisymbrium irio, a weed of family Brassicaceae, were tested for evaluation of their antifungal activity against M. phaseolina. In screening bioassays, antifungal activity of methanolic extracts of 1% to 6% concentrations of different parts (leaf, stem, root and fruit) of the weed was assessed against the fungal pathogen. Methanolic leaf and root extracts significantly reduced fungal biomass up to 59% and 69% over control, respectively. Methanolic leaf and root extracts were further partitioned using four organic solvents namely n-hexane, chloroform, ethyl acetate and n-butanol in order of increasing polarity. Antifungal activity of different concentrations of these fractions (3.125, 6.25, …, 200 mg mL-1) was assessed against the pathogen. Chloroform and n-hexane fractions of methanolic leaf extract showed highly pronounced activity resulting in 35-75% and 15-87% reduction in fungal biomass over corresponding control treatments, respectively. Likewise, the highest concentration (200 mg mL-1) of chloroform, ethyl acetate and n-butanol fractions of methanolic root extract reduced fungal biomass by 75%, 70% and 87%, respectively. The present study concludes that chloroform and n-butanol fractions of methanolic leaf and root extracts of S. irio, respectively, contain potent antifungal constituents for management of M. phaseolina.

Effect of yeasts on biodegradation potential of immobilized cultures of white rot fungi

The aim was to investigate the effect of yeast organisms on the degradation process by immobilized cultures of ligninolytic fungi. Immobilization was accomplished by 7-day colonization of polyamide mesh with mycelial fragments. Irpex lacteus decolorized >90% of the initial concentration of 150mgl−1 of anthraquinone Remazol Brilliant Blue R dye in three subsequent decolorization cycles and the degradation capacity was not negatively affected by the presence of 106Saccharomyces cerevisiae cells per ml in the mixed culture. The yeast was not able to degrade the dye. I. lacteus biofilm was also resistant to bacterial infection with E. coli. Inoculation of the yeast to pre-formed I. lacteus biofilm culture resulted in a reduction of fungal biomass by 27%. Levels of LiP, MnP and laccase of I. lacteus were not much influenced by S. cerevisiae or E. coli. Similar resilience of P. ostreatus biofilms was observed after exposure to yeast Issatchenkia occidentalis when the fungal degradation capacity measured with Reactive Orange 16 azo dye was maintained over two decolorization cycles. I. occidentalis did not degrade the dye under the conditions used. Formation of densely packed fungal biofilms with abundant extracellular polysaccharide was not impeded by the yeast. Increase of MnP and laccase levels attributable to the presence of I. occidentalis was observed.

ANTIFUNGAL ACTIVITY OF Cirsium arvense EXTRACTS AGAINST PHYTOPATHOGENIC FUNGUS Macrophomina phaseolina

ABSTRACT Leaves, stems, roots and inflorescence of the asteraceous weed Circium arvense were extracted in methanol for two weeks. Methanol was evaporated in a rotary evaporator. Different concentrations (1, 2, 3, 4 and 5%) of methanolic extracts were prepared, and their antifungal activities were studied against Macrophomina phaseolina, using malt extract broth as growth medium. In general, extracts of all plant parts showed antifungal activities to variable extents. The highest antifungal activity occurred due to methanolic extract of leaves, followed by stem and root extracts, resulting in 10-74%, 6-57% and 11-39% reduction in fungal biomass over control, respectively. Inflorescence extract showed the least antifungal activity, resulting in 2-30% reduction in fungal biomass over control. There was a linear and inverse relationship between extract concentrations and fungal biomass for extracts of all the four parts. GC-MS analysis showed that there were 10 compounds in most effective methanolic leaf extract. Among these, 10-octadecanoic acid, methyl ester (26.442%), 2H-1-benzopyran, 6,7-dimethoxy-2-2-dimethyl (20.195%), hexadecanoic acid, methyl ester (15.752%) and 9,12-octadecadienoic acid (Z,Z)-, methyl ester (12.628%) were predominant compounds in the extract that may be responsible for antifungal activity. This study concludes that methanolic leaf extracts of C. arvense can be used for the management of M. phaseolina.

FUNGICIDAL POTENTIAL OF ALLELOPATHIC WEED Cenchrus pennisetiformis ON GROWTH OF Fusarium oxysporum f. sp. lycopersici UNDER CHROMIUM STRESS

ABSTRACT The present study was conducted to assess antifungal potential of the allelopathic grass Cenchrus pennisetiformis Hochst. & Steud. against the fungal plant pathogen Fusarium oxysporum f. sp. lycopersici (cause of tomato wilt disease) under chromium stress. Laboratory experiments were performed in 10 mL volume glass test tubes each containing 1.0 mL of malt extract broth with seven concentrations (0, 50, 100, 150, 200, 250, 300 and 350 ppm) of each of Cr(III) and Cr(VI), and two concentrations (5% and 6%) of methanolic leaf, stem or root extract of C. pennisetiformis. A metal + weed extract amended medium was inoculated with the pathogen and incubated for 7 days at 25 C. Different concentrations of Cr(III) and Cr(VI) proved equally inhibitory resulting in 10-84% and 18-89% reduction in fungal biomass, respectively. Methanolic leaf, stem and root extracts of the weed reduced fungal biomass by 12-25%, 14-23% and 46-50%, respectively, over negative control. In combined application of methanolic extracts of different parts of C. pennisetiformis and metal solutions, root extract in combination with either Cr(III) or Cr(VI) showed the highest inhibitory potential against the fungus followed by leaf and stem extracts. In combination with methanolic root, leaf and stem extracts, different concentrations of Cr(III) and Cr(VI) significantly reduced fungal biomass by 54-99%, 14-99% and 9-95%, respectively, over negative control. Such studies have not been carried out previously. Results of the present investigation suggest that F. oxysporum oxysporum f. sp. lycopersici, the cause of Fusarium wilt disease in tomato, can be managed by application of extracts (or alternatively biomass) of C. pennisetiformis in chromium contaminated soils.

Are nitrate production and retention processes in subtropical acidic forest soils responsive to ammonium deposition?

Changes in soil N-cycling and retention processes in subtropical/tropical acidic forest ecosystems under anthropogenic N inputs are not well understood. We conducted a laboratory 15N tracing study on an acid soil (pH values: 4.6 to 5.0) from a subtropical forest fertilized for more than 2.5 years at a rate of 0, 40, and 120 kg NH4ClN ha−1 yr−1, respectively. To get a better resolution of mechanistic changes in soil N cycling and retention processes under NH4+ additions, we used a conceptual 15N tracing model to quantify process-specific and pool-specific N transformation rates in soils. Gross N mineralization rates decreased at high NH4+ additions, which were paralleled by a reduction in fungal biomass and mineralization of recalcitrant organic N. Gross NH4+ immobilization rates did not show a change with increasing NH4+ additions. Interestingly, soil NO3− production (heterotrophic, autotrophic, and gross nitrification) and retention (NO3− immobilization and dissimilatory nitrate reduction to ammonium) showed insensitivity to increasing additions of NH4+. The mechanisms behind the lack of response of heterotrophic nitrification were unclear, but possibly related to the absence of significant changes in soil C: N ratio and soil acidity under increased NH4+ additions. Because of the low autotrophic nitrification potential and the lack of NH4+ limitation to autotrophic nitrifiers, autotrophic nitrification was unresponsive to NH4+ additions. NO3− immobilization rates appeared to be controlled by the NO3− produced from heterotrophic nitrification, as indicated by the positive relationship between NO3− immobilization and heterotrophic nitrification (R2 = 0.59, p = 0.015), thus showing a lack of a change under increased NH4+ additions. DNRA seemed to be inherently less responsive to environmental changes such as NH4+ deposition. Our work demonstrates that enhanced NH4+ deposition has a low potential to stimulate soil NO3− production and weaken soil retention of NO3− in this, and perhaps other subtropical/tropical acidic forest ecosystems.

ANTIFUNGAL ACTIVITY OF METHANOLIC ROOT EXTRACT OF WITHANIA SOMMNIFERA AGAINST FUSARIUM OXYSPORUM F. SP. CEPAE

Background: Basal rot of onion (Allium cepa L.) caused by Fusarium oxysporum f. sp. cepae is a common soil-borne disease that causes significant yield losses. Generally, synthetic fungicides are used to combat the menace which cause environmental pollution. The present study was carried out to assess the antifungal activity of Withania somnifera (L.) Dunal, a Solanaceous medicinal plant, against the pathogen of this disease. 
 Materials and Methods: Different concentrations (from 0.5 to 4%) of methanolic extract of root, stem and fruit of the test plant species were prepared and their bioactivity was assessed against the target fungal pathogen. Methanolic extract of root was further fractionated with n-hexane, chloroform, ethyl acetate and n-butanol. Different concentrations of these extracts viz. 200, 100… 3.125 mg mL-1 were prepared and assessed for their antifungal activities.
 Results: Methanolic root extract exhibited the best antifungal activity causing up to 93% decrease in biomass of the fungal pathogen. n-hexane, chloroform and ethyl acetate fractions of methanolic root extract exhibited pronounced antifungal activity resulting in 46–79%, 40–73% and 35–76% reduction in fungal biomass, respectively. 
 Conclusion: The present study concludes that root extract of W. somnifera possesses potent antifungal constituents which can be used for the control of F. oxysporum f. sp. cepae.

Physiological and morphological changes in Botrytis cinerea induced by the plant alkaloid camptothecin

Botrytis cinerea causes diseases in plants that lead to severe crop losses. This study aimed to evaluate the physiological and morphological changes and gene expression induced by camptothecin, a monoterpene indole alkaloid, for the control of B. cinerea. Using a single concentration of 8.0 μg/mL, which showed to reduce 50 % of the dry weight of fungal mycelia, the alkaloid caused a 25 to 30 % reduction in colony diameter from 24 to 72 h of incubation and 34 to 54.7 % reduction in fungal biomass, from 24 to 96 h of incubation. Furthermore, changes in mycelial morphology were noticed based on both scanning and transmission electron microscopic observations. POD activity was reduced by 49.1 %, but PAL and SOD activity were not affected. Moreover, q-PCR analysis showed that the expression of erg27, os1, and SDHB were not affected, but the expression of cytb increased significantly, thus suggesting the involvement of this respiration-related gene in the response of B. cinerea to camptothecin. In summary, the alkaloid has the potential to improve the control of gray mold caused by B. cinerea.

Inhibitory effect of essential oils on Aspergillus ochraceus growth and ochratoxin A production.

Ochratoxin A (OTA) is a mycotoxin which is a common contaminant in grains during storage. Aspergillus ochraceus is the most common producer of OTA. Essential oils play a crucial role as a biocontrol in the reduction of fungal contamination. Essential oils namely natural cinnamaldehyde, cinnamon oil, synthetic cinnamaldehyde, Litsea citrate oil, citral, eugenol, peppermint, eucalyptus, anise and camphor oils, were tested for their efficacy against A. ochraceus growth and OTA production by fumigation and contact assays. Natural cinnamaldehyde proved to be the most effective against A. ochraceus when compared to other oils. Complete fungal growth inhibition was obtained at 150–250 µL/L with fumigation and 250–500 µL/L with contact assays for cinnamon oil, natural and synthetic cinnamaldehyde, L. citrate oil and citral. Essential oils had an impact on the ergosterol biosynthesis and OTA production. Complete inhibition of ergosterol biosynthesis was observed at ≥100 µg/mL of natural cinnamaldehyde and at 200 µg/mL of citral, but total inhibition was not observed at 200 µg/mL of eugenol. But, citral and eugenol could inhibit the OTA production at ≥75 µg/mL and ≥150 µg/mL respectively, while natural cinnamaldehyde couldn’t fully inhibit OTA production at ≤200 µg/mL. The inhibition of OTA by natural cinnamaldehyde is mainly due to the reduction in fungal biomass. However, citral and eugenol could significant inhibit the OTA biosynthetic pathway. Also, we observed that cinnamaldehyde was converted to cinnamic alcohol by A. ochraceus, suggesting that the antimicrobial activity of cinnamaldehyde was mainly attributed to its carbonyl aldehyde group. The study concludes that natural cinnamaldehyde, citral and eugenol could be potential biocontrol agents against OTA contamination in storage grains.