Plant Pathogen Rhizoctonia Solani Research Articles

Functional characterization of the AGL1 aegerolysin in the mycoparasitic fungus Trichoderma atroviride reveals a role in conidiation and antagonism

Aegerolysins are small secreted pore-forming proteins that are found in both prokaryotes and eukaryotes. The role of aegerolysins in sporulation, fruit body formation, and in lysis of cellular membrane is suggested in fungi. The aim of the present study was to characterize the biological function of the aegerolysin gene agl1 in the mycoparasitic fungus Trichoderma atroviride, used for biological control of plant diseases. Gene expression analysis showed higher expression of agl1 during conidiation and during growth in medium supplemented with cell wall material from the plant pathogenic fungus Rhizoctonia solani as the sole carbon source. Expression of agl1 was supressed under iron-limiting condition, while agl1 transcript was not detected during T. atroviride interactions with the prey fungi Botrytis cinerea or R. solani. Phenotypic analysis of agl1 deletion strains (Δagl1) showed reduced conidiation compared to T. atroviride wild type, thus suggesting the involvement of AGL1 in conidiation. Furthermore, the Δagl1 strains display reduced antagonism towards B. cinerea and R. solani based on a secretion assay, although no difference was detected during direct interactions. These data demonstrate the role of AGL1 in conidiation and antagonism in the mycoparasitic fungus T. atroviride.

An electrochemical advanced oxidation process (EAOP) for the inactivation ofRhizoctonia solaniin fertigation solutions

Boron-doped diamond anodes and stainless-steel cathodes were employed in an electrochemical flow cell and evaluated for inactivation efficacy on the plant pathogen Rhizoctonia solani J.G. Kühn [Thanatephorus cucumeris (A.B. Frank) Donk] in hydroponic fertigation water. The electrochemical system showed promise as an electrochemical advanced oxidation process (EAOP) in that significant reductions in R. solani (AG-8) were achieved; however, to achieve complete inactivation the system required supplemental chloride (20 mg L−1), while using 9.09 mA cm−2. The chloride allowed for low levels of free chlorine (≤0.17 mg L−1) that were more active in the bulk solution, complementing the electrode surface EAOP reactions. Perchlorate production was an initial concern but was found to be negligible under the conditions tested. Small but significant increases in nitrate, ammonium, and sulphate were observed following treatment. These increases are hypothesized to originate from the degradation of proteins and amino acids released during pathogen cell disruption.

Chemical Composition of an Aphid Antifeedant Extract from an Endophytic Fungus, Trichoderma sp. EFI671.

Botanical and fungal biopesticides, including endophytes, are in high demand given the current restrictive legislations on the use of chemical pesticides. As part of an ongoing search for new biopesticides, a series of fungal endophytes have been isolated from selected medicinal plants including Lauraceae species. In the current study, an extract from the endophytic fungus Trichoderma sp. EFI 671, isolated from the stem parts of the medicinal plant Laurus sp., was screened for bioactivity against plant pathogens (Fusarium graminearum, Rhizoctonia solani, Sclerotinia sclerotiorum and Botrytis cinerea), insect pests (Spodoptera littoralis, Myzus persicae, Rhopalosiphum padi) and plant parasites (Meloidogyne javanica), with positive results against M. persicae. The chemical study of the neutral fraction of the active hexane extract resulted in the isolation of a triglyceride mixture (m1), eburicol (2), β-sitostenone (3), ergosterol (4) and ergosterol peroxide (5). The free fatty acids present in the acid fraction of the extract and in m1 (oleic, linoleic, palmitic and stearic) showed strong dose-dependent antifeedant effects against M. persicae. Liquid (potato dextrose broth, PDB and Sabouraud Broth, SDB) and solid (corn, sorghum, pearl millet and rice) growth media were tested in order to optimize the yield and bioactivity of the fungal extracts. Pearl millet and corn gave the highest extract yields. All the extracts from these solid media had strong effects against M. persicae, with sorghum being the most active. Corn media increased the methyl linoleate content of the extract, pearl millet media increased the oleic acid and sorghum media increased the oleic and linoleic acids compared to rice. The antifeedant effects of these extracts correlated with their content in methyl linoleate and linoleic acid. The phytotoxic effects of these extracts against ryegrass, Lolium perenne, and lettuce, Lactuca sativa, varied with culture media, with sorghum being non- toxic.

Upcycling Phosphorus Recovered from Anaerobically Digested Dairy Manure to Support Production of Vegetables and Flowers

Dissolved air flotation (DAF) separates phosphorus (P)-rich fine solids from anaerobically digested dairy manure, creating opportunities to export surplus P to the marketplace as a bagged plant food product. Seedlings of tomato and marigold were amended at various volume per volume (v/v) ratios with plant foods consisting of fine solids upcycled (i.e., transformed into a higher quality product) by drying and blending with other organic residuals. A plate competition assay was conducted to assess the fine solids’ potential to suppress the plant pathogen Rhizoctonia solani. Plant foods were comprised of 2.0–2.1% N, 0.8–0.9% P and 0.6–0.8% K. Extractions indicated that plant foods contained a mixture of plant-available and slow-release P. At 6% v/v plant food, dry biomass of marigold and tomato were six-times greater than the unamended control and not significantly different from a market alternative treatment. Fine solids exhibited negligible potential to suppress R. solani. This study indicates that DAF-separated fine solids could be used to support horticulture, providing information for design of a circular economy approach to dairy manure nutrient management. Life cycle assessment and business model development for this nutrient recovery strategy are necessary next steps to further guide sustainability efforts.

Activity of plant extracts/essential oils against three plant pathogenic fungi and mosquito larvae: GC/MS analysis of bioactive compounds

Certain natural products extracted from different parts of medicinal and aromatic plants were examined for their antifungal activity against three plant pathogenic fungi, Fusarium oxysporum, Rhizoctonia solani, and Alternaria solani, and insecticidal activity against mosquito larvae (Culex pipiens). Acetone extract of Tectona grandis showed the highest antifungal activity against R. solani and A. solani with EC50 values of 118 and 294 μg/mL, respectively. The highest larvicidal activity was displayed by the essential oils of Ocimum basilicum and Eucalyptus gomphocephala with LC50 value of 22, and 30 mg/L, respectively. By gas chromatography–mass spectrometry (GC/MS) analysis 3-allylguaiacol (65.8%) and eugenol acetate (46.6%) were the main compounds in Syzygium aromaticum methanolic extract and essential oil, respectively. The main compound in T. grandis acetone extract was cyclohexylpentyl oxalate (8.7%); its water extract contained (E)-4,4-dimethyl-2-pentene (51.1%); E. gomphocephala branch oil contained p-cymene (28.8%); Euphorbia paralias leaf extract contained 1βH-romneine (26.3%); the seed extract contained α-linolenic acid, TMS (15.2%); Punica granatum extract contained furfural (32.1%); and O. basilicum essential oil contained estragole (65.9%). Thus, extracts from the tested plants can be used as natural biofungicides to manage diseases caused by F. oxysporum, R. solani, and A. solani. Additionally, these extracts show potential larvicide activities against mosquito larvae.

ISOLATION AND IDENTIFICATION OF TRICHODERMA SPECIES FROM SOILS FOR USING AGAINST SOME SOILBORNE FUNGAL PATHOGENS

Hydrolytic enzyme producing Trichoderma species have long been recognized as an agent for controlling plant diseases caused by various phytopathogenic fungi. A study was done to characterize four isolates of Trichoderma isolated from the rhizosphere of different crop plants to characterize and identify certain biocontrol related enzymes (β1,3-glucanase and chitinase). Morphological and molecular characterize action of antagonistic ability of Trichoderma species were studied. On the basis of morphological and culture characteristics, the Trichoderma isolates were identified as T. harizanum (1 isolate), T. viride (3 isolates). These isolates were tested against plant pathogens (Rhizoctonia solani, Pythium and Fusarium oxysporum). The best isolate producing chitinase (overproduction) was T. harizanum from (160 to 0.64) mg/ml. On the other hand the best isolate gave over production β-1; 3-glucanase over production was T. viride from (2250 to 2.20 mg/ml)

Efficacy of electrolytically-derived disinfectant against dispersal of Fusarium oxysporum and Rhizoctonia solani in hydroponic tomatoes

Abstract Demand for conservation and recycling of water has increased significantly. Therefore irrigation water used for horticultural or agricultural purposes needs to be treated before being reused to eradicate plant pathogens and thereby reducing the risk of pathogen dispersal and losses due to disease. The economically important fungal plant pathogens Fusarium oxysporum (Synder and Hans) and Rhizoctonia solani (Kuhn) were selected to examine the efficacy of nutrient solution treatment by electrolytic disinfection to prevent the dispersal of these pathogens in the hydroponic production of tomatoes (Solanum lycopersicum Mill.). First, we determined the efficacy of the disinfectant to inactivate F. oxysporum and R. solani in vitro. The electrolytically generated potassium hypochlorite (KClO) was tested at five concentrations of free chlorine (0.2, 0.5, 0.8, 1.0, 2.0 mg/L) in nutrient solutions of pH 5.5, 6.0 and 6.5 with four contact times (5, 30, 60, 120 min). Best sanitation was achieved in nutrient solution at pH 6.0. In vitro, F. oxysporum required 2 mg/L at 30 min for complete inactivation whereas chlorination had only a minimal effect on viability of R. solani. Subsequent trials under practical conditions applied the disinfectant via a new sensor-based disinfection procedure. Potassium hypochlorite solution produced on site and injected into a recirculating nutrient solution once a week for 60 min at a free chlorine concentration of 0.5 mg/L (ORP 780 mV) inhibited the dispersal of F. oxysporum and R. solani during the entire test period of 16 weeks. In contrast all tomato test plants irrigated with untreated nutrient solution became infected with F. oxysporum and a third of them additionally with R. solani. At the applied dose no plant damage occurred. Thus, the treatment proved to be effective and applicable to prevent dispersal of fungal pathogens by nutrient solution under simulated field conditions.

In-Vitro Inhibition of Pythium ultimum, Fusarium graminearum, and Rhizoctonia solani by a Stabilized Lactoperoxidase System alone and in Combination with Synthetic Fungicides

Advances in enzyme stabilization and immobilization make the use of enzymes for industrial applications increasingly feasible. The lactoperoxidase (LPO) system is a naturally occurring enzyme system with known antimicrobial activity. Stabilized LPO and glucose oxidase (GOx) enzymes were combined with glucose, potassium iodide, and ammonium thiocyanate to create an anti-fungal formulation, which inhibited in-vitro growth of the plant pathogenic oomycete Pythium ultimum, and the plant pathogenic fungi Fusarium graminearum and Rhizoctonia solani. Pythium ultimum was more sensitive than F. graminearum and R. solani, and was killed at LPO and GOx concentrations of 20 nM and 26 nM, respectively. Rhizoctonia solani and F. graminearum were 70% to 80% inhibited by LPO and GOx concentrations of 242 nM and 315 nM, respectively. The enzyme system was tested for compatibility with five commercial fungicides as co-treatments. The majority of enzyme + fungicide co-treatments resulted in additive activity. Synergism ranging from 7% to 36% above the expected additive activity was observed when P. ultimum was exposed to the enzyme system combined with Daconil® (active ingredient (AI): chlorothalonil 29.6%, GardenTech, Lexington, KY, USA), tea tree oil, and mancozeb at select fungicide concentrations. Antagonism was observed when the enzyme system was combined with Tilt® (AI: propiconazole 41.8%, Syngenta, Basel, Switzerland) at one fungicide concentration, resulting in activity 24% below the expected additive activity at that concentration.

Synthesis and characterization of chitosan\u2013copper nanocomposites and their fungicidal activity against two sclerotia-forming plant pathogenic fungi

In this report, the metal-vapor synthesis (MVS) was used for the preparation of copper nanoparticles which was then used for the preparation of chitosan–copper nanocomposite. The antifungal activity of Cu@Chit NCs against two sclerotium-forming plant pathogenic fungi Sclerotium rolfsii (S. rolfsii) and Rhizoctonia solani (R. solani) AG-4 was evaluated in vitro and their effects on hyphal morphology, and sclerotia formation were observed for the first time. The NCs were prepared through impregnation of chitosan with colloid solution of copper nanoparticles in organic solvent (acetone or toluene). Transmission electron microscopy shows that the particles have predominantly spherical form, polydisperse character, the mean diameter about 2–3 nm and a rather uniform distribution in the chitosan matrice. Analysis of the small angle scattering curves suggests that the copper particles in the NCs with the size of ≤2 nm are mostly located in the chitosan pores with the same size. The effect of Cu@Chit NCs on fungal growth reveals some significant inhibitory activity against two tested fungi. The highest level of inhibition against S. rolfsii and R. solani AG-4 was observed using the high concentrations of Cu@Chit NC prepared using acetone as a solvent. A loss of the cytoplasm content, cytoplasmic coagulation, irregular shape of mycelia, or destruction in the hyphae was confirmed. The experiments demonstrate that the Cu@Chit NC synthesized via MVS using acetone was more effective than that of toluene in inhibiting fungal hyphae growth against R. solani AG-4 and S. rolfsii. The results show that the Cu@Chit NCs are fungicidal against both the tested fungus at high concentrations and the fungicidal or fungistatic activity is dependent on the tested fungus species.

Genes of the de novo and Salvage Biosynthesis Pathways of Vitamin B6 are Regulated under Oxidative Stress in the Plant Pathogen Rhizoctonia solani.

Vitamin B6 is recognized as an important cofactor required for numerous metabolic enzymes, and has been shown to act as an antioxidant and play a role in stress responses. It can be synthesized through two different routes: salvage and de novo pathways. However, little is known about the possible function of the vitamin B6 pathways in the fungal plant pathogen Rhizoctonia solani. Using genome walking, the de novo biosynthetic pathway genes; RsolPDX1 and RsolPDX2 and the salvage biosynthetic pathway gene, RsolPLR were sequenced. The predicted amino acid sequences of the three genes had high degrees of similarity to other fungal PDX1, PDX2, and PLR proteins and are closely related to other R. solani anastomosis groups. We also examined their regulation when subjected to reactive oxygen species (ROS) stress inducers, the superoxide generator paraquat, or H2O2, and compared it to the well-known antioxidant genes, catalase and glutathione-S-transferase (GST). The genes were differentially regulated with transcript levels as high as 33 fold depending on the gene and type of stress reflecting differences in the type of damage induced by ROS. Exogenous addition of the vitamers PN or PLP in culture medium significantly induced the transcription of the vitamin B6 de novo encoding genes as early as 0.5 hour post treatment (HPT). On the other hand, transcription of RsolPLR was vitamer-specific; a down regulation upon supplementation of PN and upregulation with PLP. Our results suggest that accumulation of ROS in R. solani mycelia is linked to transcriptional regulation of the three genes and implicate the vitamin B6 biosynthesis machinery in R. solani, similar to catalases and GST, as an antioxidant stress protector against oxidative stress.

Transcriptomic changes in the plant pathogenic fungus Rhizoctonia solani AG-3 in response to the antagonistic bacteria Serratia proteamaculans and Serratia plymuthica.

BackgroundImproved understanding of bacterial-fungal interactions in the rhizosphere should assist in the successful application of bacteria as biological control agents against fungal pathogens of plants, providing alternatives to chemicals in sustainable agriculture. Rhizoctonia solani is an important soil-associated fungal pathogen and its chemical treatment is not feasible or economic. The genomes of the plant-associated bacteria Serratia proteamaculans S4 and Serratia plymuthica AS13 have been sequenced, revealing genetic traits that may explain their diverse plant growth promoting activities and antagonistic interactions with R. solani. To understand the functional response of this pathogen to different bacteria and to elucidate whether the molecular mechanisms that the fungus exploits involve general stress or more specific responses, we performed a global transcriptome profiling of R. solani Rhs1AP anastomosis group 3 (AG-3) during interaction with the S4 and AS13 species of Serratia using RNA-seq.ResultsApproximately 104,504 million clean 75-100 bp paired-end reads were obtained from three libraries, each in triplicate (AG3-Control, AG3-S4 and AG3-AS13). Transcriptome analysis revealed that approximately 10 % of the fungal transcriptome was differentially expressed during challenge with Serratia. The numbers of S4- and AS13-specific differentially expressed genes (DEG) were 866 and 292 respectively, while there were 1035 common DEGs in the two treatment groups. Four hundred and sixty and 242 genes respectively had values of log2 fold-change > 3 and for further analyses this cut-off value was used. Functional classification of DEGs based on Gene Ontology enrichment analysis and on KEGG pathway annotations revealed a general shift in fungal gene expression in which genes related to xenobiotic degradation, toxin and antioxidant production, energy, carbohydrate and lipid metabolism and hyphal rearrangements were subjected to transcriptional regulation.ConclusionsThis RNA-seq profiling generated a novel dataset describing the functional response of the phytopathogen R. solani AG3 to the plant-associated Serratia bacteria S4 and AS13. Most genes were regulated in the same way in the presence of both bacterial isolates, but there were also some strain-specific responses. The findings in this study will be beneficial for further research on biological control and in depth exploration of bacterial-fungal interactions in the rhizosphere.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1758-z) contains supplementary material, which is available to authorized users.

Antibiosis functions during interactions of Trichoderma afroharzianum and Trichoderma gamsii with plant pathogenic Rhizoctonia and Pythium.

Trichoderma afroharzianum is one of the best characterized Trichoderma species, and strains have been utilized as plant disease suppressive inoculants. In contrast, Trichoderma gamsii has only recently been described, and there is limited knowledge of its disease suppressive efficacies. Comparative studies of changes in gene expression during interactions of these species with their target plant pathogens will provide fundamental information on pathogen antibiosis functions. In the present study, we used complementary DNA amplified fragment length polymorphism (cDNA-AFLP) analysis to investigate changes in transcript profiling of T. afroharzianum strain LTR-2 and T. gamsii strain Tk7a during in vitro interactions with plant pathogenic Rhizoctonia solani and Pythium irregulare. Considerable differences were resolved in the overall expression profiles of strains LTR-2 and Tk7a when challenged with either plant pathogen. In strain LTR-2, previously reported mycoparasitism-related genes such as chitinase, polyketide synthase, and non-ribosomal peptide synthetase were found to be differentially expressed. This was not so for strain Tk7a, with the only previously reported antibiosis-associated genes being small secreted cysteine-rich proteins. Although only one differentially expressed gene was common to both strains LTR-2 and Tk7a, numerous genes reportedly associated with pathogen antibiosis processes were differentially expressed in both strains, including degradative enzymes and membrane transport proteins. A number of novel potential antibiosis-related transcripts were found from strains LTR-2 and Tk7a and remain to be identified. The expression kinetics of 20 Trichoderma (10 from strain LTR-2, 10 from strain Tk7a) transcript-derived fragments (TDFs) were quantified by quantitative reverse transcription PCR (RT-qPCR) at pre- and post-mycelia contact stages of Trichoderma-prey interactions, thereby confirming differential gene expression. Collectively, this research is providing information to elucidate the antibiosis mechanisms and disease suppressive activities of T. afroharzianum and T. gamsii against soilborne fungal and oomycete plant pathogens.

Identification of signatory secondary metabolites during mycoparasitism of Rhizoctonia solani by Stachybotrys elegans.

Stachybotrys elegans is able to parasitize the fungal plant pathogen Rhizoctonia solani AG-3 following a complex and intimate interaction, which, among others, includes the production of cell wall-degrading enzymes, intracellular colonization, and expression of pathogenic process encoding genes. However, information on the metabolome level is non-existent during mycoparasitism. Here, we performed a direct-infusion mass spectrometry (DIMS) metabolomics analysis using an LTQ Orbitrap analyzer in order to detect changes in the profiles of induced secondary metabolites of both partners during this mycoparasitic interaction 4 and 5 days following its establishment. The diketopiperazine(s) (DKPs) cyclo(S-Pro-S-Leu)/cyclo(S-Pro-S-Ile), ethyl 2-phenylacetate, and 3-nitro-4-hydroxybenzoic acid were detected as the primary response of Rhizoctonia 4 days following dual-culturing with Stachybotrys, whereas only the latter metabolite was up-regulated 1 day later. On the other hand, trichothecenes and atranones were mycoparasite-derived metabolites identified during mycoparasitism 4 and 5 days following dual-culturing. All the above secondary metabolites are known to exhibit bioactivity, including fungitoxicity, and represent key elements that determine the outcome of the interaction being studied. Results could be further exploited in programs for the evaluation of the bioactivity of these metabolites per se or their chemical analogs, and/or genetic engineering programs to obtain more efficient mycoparasite strains with improved efficacy and toxicological profiles.

Ieodoglucomide C and Ieodoglycolipid, New Glycolipids from a Marine-Derived Bacterium Bacillus licheniformis 09IDYM23.

Chemical examination of the ethyl acetate extract from the fermentation broth of the marine-derived bacterium Bacillus licheniformis resulted in the isolation of two new glycolipids, ieodoglucomide C (1) and ieodoglycolipid (2). The structural characterization of 1 and 2 was achieved by extensive spectroscopic evidence, including 2D NMR experiments. A combination of chemical derivatization techniques followed by NMR studies, LC-MS data analysis and a literature review was deployed for the establishment of the stereo-configurations of 1 and 2. Compounds 1 and 2 exhibited good antibiotic properties against Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Salmonella typhi, Escherichia coli and Pseudomonas aeruginosa with MICs ranging from 0.01 to 0.05 μM. Furthermore, the antifungal activity of 1 and 2 was evaluated against plant pathogenic fungi Aspergillus niger, Rhizoctonia solani, Botrytis cinerea and Colletotrichum acutatum as well as the human pathogen Candida albicans. Compounds 1 and 2 inhibited the mycelial growth of these pathogens with MIC values of 0.03-0.05 μM, revealing that these compounds are good candidates for the development of new fungicides.

Heterologous expression of the antifungal β-chitin binding protein CBP24 from bacillus thuringiensis and its synergistic action with bacterial chitinases.

The genome sequence analysis of Bacillus thuringiensis serovar konkukian S4 has shown to contain two chitinases (Chi74, Chi39) and two chitin-binding proteins (CBP50 and CBP24). The Chi74, Chi39 and CBP50 have been characterized previously. The chitin-binding protein CBP24 was cloned and heterologously expressed in Escherichia coli. The recombinant protein was purified using a Ni-NTA purification system. The purified protein was used to study its substrate binding activity using crystalline chitin variants as substrates. The Bmax and Kd values have shown that it preferably binds to β-type of the crystalline chitin at a range of pH with peak activity between 5.5-7.5. To elucidate the role of CBP24 in the chitin degradation system of S4, the purified chitinases Chi74, Chi39 along with the ChiA from Serratia proteamcualans were used in different combinations with the CBP24 and chitinolytic activity was assayed. It was shown that the CBP24 acts synergistically with chitin degradation activity of bacterial chitinases non-specifically. Moreover, the CBP24 has shown antifungal activity against plant pathogenic fungi Fusarium oxysporum and Rhizoctonia solani. The present study will lead us to develop a technology for environmental friendly conversion of chitin to valuable products.

In-vitro antagonism of Trichoderma spp. against Sclerotium rolfsii and Rhizoctonia solani and their response towards growth of cucumber, bottle gourd and bitter gourd

In the current study we evaluated the production of potential growth-promoting metabolites (IAA, phosphate, siderophore) for 20 isolates of Trichoderma collected from different geographical locations of Chhattisgarh and assessed their growth response on cucumber, bottle gourd and bitter gourd. All the Trichoderma isolates were able to release inorganic phosphorus from tri-calcium phosphate and showed consistent ability to produce siderophores and indole-3-acetic acid (IAA). The production of these metabolites varied greatly within species. Using the same set of isolates we evaluated the production of potential cell wall degrading enzymes. Confrontation assays of Trichoderma isolates against two soilborne plant pathogens (Scelrotium rolfsii and Rhizoctonia solani) expressed varying degrees of antagonistic responses, in-vitro antagonism being more effective against R. solani than S. rolfsii. The production of metabolites in all the Trichoderma isolates did not correlate with enhanced growth on cucumber, bottle gourd and bitter gourd, and bio control efficacy. However, one of the Trichoderma viride isolate (T14) was identified as highest producer of inorganic phosphate, IAA and siderophore exhibited high antagonistic and plant growth promoting ability. A characteristic aromatic odour resembling coconut in T14 isolate was observed which we speculate is due to 6-Pentyl pyrone (one of the best studied secondary metabolites having both antifungal and plant growth-promoting activities).

English

The study of morphology and bioefficacy of Trichoderma was undertaken to select the effective isolates against soil-borne pathogens. Fifty one (51) isolates (23 isolates of Trichoderma virens and 28 isolates of Trichoderma harzianum) were morphologically characterised based on the growth characteristics on PDA medium, the size and shape of phialides and conidia.These isolates were screened for bioefficacy against soil borne plant pathogens (Fusarium oxysporum, Rhizoctonia solani and Sclerotium rolfsii) based on their percent inhibition observed during dual culture, volatile and non-volatile methods. Eight T. virens isolates and 12 T. harzianum isolates were proven to be potential isolates against the soil-borne pathogens tested. No correlation was found between bioefficacy and morphology in both species isolates. Key words: Trichoderma virens, Trichoderma harzianum, morphology, dual culture, biocontrol and bioefficacy.

Substrate use and survival of fungal plant pathogens on maize residues at winter temperatures around freezing point

Climate scenarios predict increasing temperatures and higher precipitation rates in late autumn to early spring, both of which holding the potential to change the dynamics of plant residue decomposition and overall microbial activity in soil. In company with consequences for nutrient release patterns influences on the survival of fungal plant pathogens and their phytopathogenicity can be expected. Both, litter decomposition and pathogen survival, was analyzed in a 70-day litterbag incubation experiment. Continuous +4 °C was compared to permanent frost (−3 °C) and different freeze-thaw cycles for the decomposition of maize residues and disease potential of soil-borne plant pathogens Fusarium culmorum, Fusarium graminearum, and Rhizoctonia solani. Frost generally reduced maize residue decomposition. On the lower levels of CO2 production in the permanent or occasionally frost treatments pathogen inoculation had large effects on microbial maize use, indicating high saprotrophic activity of pathogens even in cold winter scenarios. Pathogen inoculation led to higher amino sugar contents of maize residue dwelling microbial organisms. At constant 4 °C remarkable high amounts of glucosamine were detected, indicating higher substrate use efficiency without frost. Both, temperature treatments as well as intra- and interspecific competition directed the development of pathogens after inoculation. F. culmorum took large advantage from the non-frost scenario, while no significant increase was found under continuous frost. F. graminearum was also able to increase its abundance at +4 °C. But this was strongly reduced when F. graminearum was in competition to the other two pathogens. In summary, F. culmorum was found to be highly frost tolerant and competitive against F. graminearum, particularly under conditions of freeze-thaw cycles since F. culmorum was able to take a large share of saprotrophic litter residue use under the cold conditions. Biomass of R. solani was strongly decomposed in all treatments. We conclude that constant mild conditions during winter can increase biomass of F. culmorum and F. graminearum in crop residues, causing increased infection pressure in the next season. In contrast to that, frost and freeze-thaw events can lower the build-up of Fusarium biomass and thus diminish the risk of crop infection.

Antimicrobial activity of probiotic bacteria

Probiotic strains were isolated from different cheeses (turkey and domiatii and cottage and kariesh cheese) and fermented products (yoghurt, raib, zeer milk and kishk). The isolates were screened for rate of growth increase in MSR broth at pH 3, pH4, acid production after 48 hours and degree of bile salt (0.3%) tolerance, strength of adhesion and clotting time (3 to 48 hrs) of skim milk. The good fourteen isolates identified as two Lactobacillus spp (S4b1 and S2a3), eleven Bifidobacterium spp. (RC1 b8, RC2 b1, SC1a4, RC4b2, FC1b1, RC2b4, RC4a3, LZ1a3 and LZa7) and one Streptoococcus spp. (RC2b3), were used against several human (Staphyllococcus aureus. and Eschericia coli) and plant (Rhizoctonia solani and Fusarium oxysporum) pathogens by examining their in vitro antimicrobial properties. Antibacterial activity of the good fourteen selected probiotic isolates in this test exhibited varying degrees of inhibitory activity against human pathogenic Staphyllococcus aureus. The isolates LZb8, S4b1 and RC2b3 exhibited the superior antibacterial activity with inhibition zones (I.Z.) ranged 8.3 - 8.4 mm followed by the isolates Kb2, LZa7 and Y2a5. The least activity was recorded for the isolates SCa4 and RC4b2 (I.Z.) ranged 2.3-2.5 mm. The antibacterial activity of the same probiotic isolates against human pathogenic Eschericia coli was almost similar to that obtained against S. aureus, and followed the same pattern. The isolates LZb8, S4b1 and RC2b3 possessed the highest activity, while the isolates SCa4 and RC4b2 were highly significantly the least active. It seems that the inhibitory activity of the isolates against E. coli was slightly less as compared to that obtained against S. aureus. The antifungal activity of the same14 probiotic isolates was tested against the plant pathogenic Rhizoctonia solani and Fusarium oxysporum. All probiotic isolates were highly significantly active on both fungi as compared to the control, showing % growth inhibition (%GI) ranges of 26.7-52.3 %, and 17.1 -51.2 % against the first and the latter fungi, respectively . The most active isolates against R. solani were RC4b2 (52.3 % GI), followed by both RC4b3 and RC1b8 (47.6 % GI). Also, the most active isolate against F. oxysporum was Y2a5 (51.2 GI).

Isolation and identification of antimicrobial secondary metabolites from Bacillus cereus associated with a rhabditid entomopathogenic nematode

The cell-free culture filtrate of Bacillus cereus associated with an entomopathogenic nematode, Rhabditis (Oscheius) sp., exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain six bioactive compounds. The structure and absolute stereochemistry of these compounds were determined based on extensive spectroscopic analyses (LCMS, FABMS, 1H NMR, 13C NMR, 1H −1H COSY, 1H −13C HMBC) and Marfey’s method. The compounds were identified as cyclo(D-Pro-D-Leu), cyclo(L-Pro-D-Met), cyclo (L-Pro-D-Phe), cyclo (L-Pro-L-Val), 3,5-dihydroxy-4-ethyl-trans-stilbene, and 3,5-dihydroxy-4-isopropylstilbene, respectively. Compounds recorded antibacterial activity against all four tested bacteria strains of Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. 3,5-dihydroxy-4-isopropylstilbene recorded activity only against Gram-positive bacteria while cyclo(L-Pro-L-Val) recorded no antibacterial activity. Best antibacterial activity was recorded by 3,5-dihydroxy-4-ethyl-trans-stilbene (4 μg/ml) against Escherichia coli. The six compounds recorded significant antifungal activities against five fungal strains tested (Aspergillus flavus, Candida albicans, Fusarium oxysporum, Rhizoctonia solani and Penicillium expansum) and they were more effective than bavistin, the standard fungicide. The activity of cyclo(D-Pro-D-Leu), cyclo(L-Pro-D-Met), 3,5-dihydroxy-4-ethyl-trans-stilbene, and 3,5-dihydroxy-4-isopropylstilbene against Candida albicans was better than amphotericin B. To the best of our knowledge, this is the first report of antifungal activity of the bioactive compounds against the plant pathogenic fungi Fusarium oxysporum, Rhizoctonia solani, and Penicillium expansum. We conclude that the Bacillus cereus strain associated with entomopathogenic nematode is a promising source of natural bioactive secondary metabolites which may receive great benefit as potential sources of new drugs in the agricultural and pharmacological industry.