Visualization of interactions between a pathogenic and a beneficial Fusarium strain during biocontrol of tomato foot and root rot

The soilborne fungus Fusarium oxysporum f. sp. radicis-lycopersici causes tomato foot and root rot (TFRR), which can be controlled by the addition of the nonpathogenic fungus F. oxysporum Fo47 to the soil. To improve our understanding of the interactions between the two Fusarium strains on tomato roots during biocontrol, the fungi were labeled using different autofluorescent proteins as markers and subsequently visualized using confocal laser scanning microscopy. The results were as follows. i) An at least 50-fold excess of Fo47over F. oxysporum f. sp. radicis-lycopersici was required to obtain control of TFRR. ii) When seedlings were planted in sand infested with spores of a single fungus, Fo47 hyphae attached to the root earlier than those of F. oxysporum f. sp. radicis-lycopersici. iii) Subsequent root colonization by F. oxysporum f. sp. radicis-lycopersici was faster and to a larger extent than that by Fo47. iv) Under disease-controlling conditions, colonization of tomato roots by the pathogenic fungus was significantly reduced. v) When the inoculum concentration of Fo47 was increased, root colonization by the pathogen was arrested at the stage of initial attachment to the root. vi) The percentage of spores of Fo47 that germinates in tomato root exudate in vitro is higher than that of the pathogen F. oxysporum f. sp. radicis-lycopersici. Based on these results, the mechanisms by which Fo47 controls TFRR are discussed in terms of i) rate of spore germination and competition for nutrients before the two fungi reach the rhizoplane; ii) competition for initial sites of attachment, intercellular junctions, and nutrients on the tomato root surface; and iii) inducing systemic resistance.     

Root colonization by phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 is essential for biocontrol of tomato foot and root rot

The phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 controls tomato foot and root rot caused by Fusarium oxysporum f. sp. radicislycopersici. To test whether root colonization is required for biocontrol, mutants impaired in the known colonization traits motility, prototrophy for amino acids, or production of the site-specific recombinase, Sss/XerC were tested for their root tip colonization and biocontrol abilities. Upon tomato seedling inoculation, colonization mutants of strain PCL1391 were impaired in root tip colonization in a gnotobiotic sand system and in potting soil. In addition, all mutants were impaired in their ability to control tomato foot and root rot, despite the fact that they produce wild-type levels of phenazine-1-carboxamide, the antifungal metabolite previously shown to be required for biocontrol. These results show, for what we believe to be the first time, that root colonization plays a crucial role in biocontrol, presumably by providing a delivery system for antifungal metabolites. The ability to colonize and produce phenazine-1-carboxamide is essential for control of F. oxysporum f. sp. radicis-lycopersici. Furthermore, there is a notable overlap of traits identified as being important for colonization of the rhizosphere and animal tissues.     

Rhizospheric bacteria as potential biocontrol agents against Fusarium wilt and crown and root rot diseases in tomato

The discovery of novel biocontrol agents requires the continuous scrutiny of native microorganisms to ensure that they will be useful on a regional scale. The goal of the present work was to discover novel antagonistic bacteria against Fusarium oxysporum ff. spp. lycopersici race 3 (Fol R3) and radicis-lycopersici (Forl) causing Fusarium wilt disease and Fusarium crown and root rot of tomatoes, respectively. High-throughput liquid antagonism screening of 1,875 rhizospheric bacterial strains followed by dual confrontation assays in 96-well plates was used to select bacteria exhibiting > 50% fungal growth inhibition. In a second dual confrontation assay in 10-cm Petri dishes, bacteria showing > 20% Fol R3 or Forl growth inhibition were further screened using a blood hemolysis test. After discarding β-hemolytic bacteria, a seedling antagonistic assay was performed to select five potential antagonists. A phylogenetic analysis of 16S rRNA identified one strain as Acinetobacter calcoaceticus (AcDB3) and four strains as members of the genus Bacillus (B. amyloliquefaciens BaMA26, Bacillus siamensis BsiDA2, B. subtilis BsTA16 and B. thuringiensis BtMB9). Greenhouse assays demonstrated that BsTA16 and AcDB3 were the most promising antagonists against Fol R3 and Forl, respectively. Pathogen biocontrol and growth promotion mechanisms used by these bacteria include the production of siderophores, biofilm, proteases, endoglucanases and indole acetic acid, and phosphate solubilization. These five bacteria exerted differential responses on pathogen control depending on the tomato hybrid, and on the growth stage of tomatoes. We report for the first time the use of an Acinetobacter calcoaceticus isolate (AcDB3) to control Forl in tomato under greenhouse conditions.     

Characterisation of the early events in atypical tomato root colonisation by a biocontrol agent, Pythium oligandrum.

The specific oomycete-plant relationship established between a biological agent, Pythium oligandrum, and tomato (Lycopersicon esculentum Mill.) plants was examined over the first 48 h after inoculation of tomato roots with the antagonist. One of the most significant effects was the quick colonisation of cortical and vascular root areas by P. oligandrum (until 9 h post-inoculation); it was similar to invasions by the major pathogens of Pythium genus, and much faster than those by Pythium-minor pathogens. Despite the multiplication of hyphae in the root areas, fungal colonisation was associated with neither host wall disruption nor host cell alterations. The colonising hyphae looked healthy till the ninth hour after inoculation, then, they progressively became highly vacuolated. Cytological observations showed that, over the first 14 h of experiment, oomycete invasion was accompanied with rare host-induced defence reactions. Biochemical analysis evidenced an accumulation of phenolic compounds starting 3 h after inoculation. The 14th hour corresponded to the beginning of rishitin (phytoalexin) synthesis. Accumulation of biochemical host defence compounds was concomitant with early signs of hyphae alterations. During the next 34 h several host reactions were regularly amplified as evidenced by the plugging of invaded host cells with heterogeneous osmiophilic or high electron-dense (ED) materials. Fungal cell decay was accompanied with the formation of oogonia in the cortex, vascular parenchyma and xylem vessels. All these early events suggest a peculiar relationship established between P. oligandrum and the plant.     

Potential of endophytic Streptomyces spp. for biocontrol of Fusarium root rot disease and growth promotion of tomato seedlings

Sixteen endophytic actinobacteria isolated from roots of native plants were evaluated for their antagonistic potential against soil-borne phytopathogenic fungi. Among them, three strong antagonistic isolates were selected and characterised for in vitro plant-growth-promoting and biocontrol traits, including production of hydrogen cyanide, indole-3-acetic acid and siderophores, chitinase and β-1,3-glucanase activities, and inorganic phosphate solubilisation. In all trials, the strain Streptomyces sp. SNL2 revealed promising features. The selected actinobacteria were investigated for the biocontrol of Fusarium oxysporum f. sp. radicis lycopersici and for growth promotion of tomato (Solanum lycopersicum L. cv. Aïcha) seedlings in autoclaved and non-autoclaved soils. All seed-bacterisation treatments significantly reduced the root rot incidence compared to a positive control (with infested soil), and the isolate SNL2 exhibiting the highest protective activity. It reduced the disease incidence from 88.5% to 13.2%, whereas chemical seed treatment with Thiram® provided 14.6% disease incidence. Furthermore, isolate SNL2 resulted in significant increases in the dry weight, shoot and root length of seedlings. 16S rDNA sequence analysis showed that isolate SNL2 was related to Streptomyces asterosporus NRRL B-24328^T (99.52% of similarity). Its interesting biocontrol potential and growth enhancement of tomato seedlings open up attractive uses of the strain SNL2 in crop improvement.     

Brown root rot of tomato, and its control, in Crete

Symptoms similar to those regarded as typical of brown root rot disease have been noticed in tomato and eggplant in Crete. Infected plants showed symptoms only during the winter. Three fungi Pyrenochaeta lycopersici, Colletotrichum coccodes and Fusarium oxysporum were consistently isolated from infected tomato roots and proved to be pathogenic to young tomato seedlings. Pycnidia of P. lycopersici were produced when small pieces of infected young tomato roots were plated out on tomato juice agar (Oxoid) and incubated at 23 °C under continuous white light. Good control of the disease was achieved when soil was fumigated with dazomet 98% (70g/m²), methyl isothiocyanate (MIC) 20%+ dichloropropane-dichloropropene mixture (DD) 80% (-Di-Trapex; 50 ml/m²), methyl bromide (75 g/m²), and vapam (200 ml/m²) + ethoprophos 10% (10 g/m²) or when solarisation was applied (soil covered by 50 μm transparent polyethylene sheet) during the whole period of August and September. Dazomet (30 g/m²) or ethoprophos 10% (10 g/m²) when combined with solarisation did not improve its effectiveness. MIC 20%+ DD 80% emulsified (Di-Trapex e.c, 50 ml/m²) was ineffective.     

Characterisation of antagonistic Bacillus and Pseudomonas strains for biocontrol potential and suppression of damping‐off and root rot diseases

Novel strains of rhizobacteria, Pseudomonas fluorescens Pf 9A‐14, Pseudomonas sp. Psp. 8D‐45 and Bacillus subtilis Bs 8B‐1, showed broad‐spectrum antagonistic activity and provided suppression of Pythium damping‐off and root rot of cucumber. Their biocontrol potential was further investigated for suppression of additional seedling diseases of cucumber (Phytophthora capsici) and radish (Rhizoctonia solani). Bacterial strains were also characterised for production of antibiotics, metabolites, volatiles, phytohormones and lytic enzymes. Seed and pre‐plant applications of all three antagonistic bacteria as cell suspension and talc or irradiated peat formulations to the infested potting mix provided overall high level of suppression of Phytophthora damping‐off and root rot of cucumber (66–85% healthy seedlings) and relatively low level of suppression of Rhizoctonia damping‐off of radish (18–38% healthy seedlings). Bacterial treatments also resulted in higher plant fresh masses. Seed coating with irradiated peat formulation of a mixture of three bacteria resulted in superior control of Phytophthora damping‐off and root rot of cucumber and much higher plant fresh masses. The presence of genes for biosynthesis of phenazine‐1‐carboxylic acid, 2,4‐diacetylphloroglucinol, pyrrolnitrin and pyoluteorin was confirmed in Pseudomonas strains, and that of fengycin, bacillomycin, bacilysin, surfactin and iturin A in B. subtilis Bs 8B‐1. All three strains produced HCN, salicylic acid, indole‐3‐acetic acid, protease and β‐1,3‐glucanase. Both Pseudomonas strains produced siderophores and only P. fluorescens Pf 9A‐14 showed phosphate solubilisation and chitinase activity. All three strains inhibited pathogen growth by producing volatiles, and gas chromatography–mass spectrometry analysis revealed eight compounds in Pf 9A‐14, 10 in Bs 8B‐1 and 4 in Psp 8D‐45, some with known antifungal activity. The antagonistic and plant‐growth promotion activities of these strains might be due to production of antibiotics, metabolites, lytic enzymes or phytohormones.     

Evaluation of fungicides and biocontrol products for the control of Phytophthora root rot of hydrangeas

Abstract

Phytophthora root rot is one of the most important diseases in almost all hydrangeas of nursery production. In this study, the efficacy of fungicides and biocontrol products against Phytophthora root rot of hydrangea was assessed in greenhouse and field experiments. Treatments used in field or greenhouse experiments were RootShield PLUS⁺, MBI110, IT-5103, Grotab, OxiPhos, TerraClean 5.0?+?TerraGrow program, Segovis, Pageant Intrinsic, Empress Intrinsic and Subdue Maxx. Pots/plots were inoculated with Phytophthora nicotianae grown on rice grains, sterilised rice grains were used for negative controls. After the trials, plant growth data (total plant weight, root weight, plant height, plant width) were recorded, and roots were assessed for disease severity using a scale of 0–100%. The treatments most effective in reducing Phytophthora root rot severity were Segovis, Empress Intrinsic, Subdue Maxx, TerraClean 5.0?+?TerraGrow program in both greenhouse and field experiments. This study will help nursery producers make proper management decisions by using recommended fungicides and biocontrol products of this study in a rotation or alone to manage Phytophthora root rot of hydrangea.     

Population dynamics of Epicoccum nigrum, a biocontrol agent against brown rot in stone fruit

Aims:  To study the population dynamics of Epicoccum nigrum on peaches and nectarines and to enhance its colonization on fruit surfaces to improve its biocontrol efficacy against brown rot.
Methods and Results:  Twelve surveys were performed to study E. nigrum populations and their effect on the number of the pathogenic Monilinia spp. conidia in peach orchards in Spain and Italy between 2002 and 2005. Fresh conidia and five different formulations of E. nigrum conidia were applied three to six times to peach and nectarine trees from full flowering to harvest. The size of the E. nigrum populations was determined from the number of colony-forming units and conidial numbers per flower or fruit. Treatment with all conidial formulations increased the size of the indigenous conidial population on peach surfaces.
Conclusions:  Formulations of E. nigrum having high viability are most effective against conidia of the pathogen when applied at pit hardening and during the month immediately before fruit harvest.
Significance and Impact of the Study:  Application of an E. nigrum conidial formulation decreased the number of conidia of Monilinia spp. on fruit surfaces during the growing season to the same extent as fungicides.     

Pseudomonas putida strain PCL1760 controls tomato foot and root rot in stonewool under industrial conditions in a certified greenhouse

Pseudomonas putida strain PCL1760 was isolated previously from the avocado rhizosphere, using an enrichment method for competitive tomato root tip colonizers that selects for biological control strains which act through the biological control mechanism “competition for nutrients and niches” (CNN). Here we demonstrate that strain PCL1760 showed significant biological control of tomato foot and root rot (TFRR), a disease caused by Fusarium oxysporum f. sp. radicis-lycopersici (Forl), in eight independent laboratory experiments in stonewool substrate. Furthermore, its activity in stonewool was also tested in an industrial setup. The presence of Forl appeared to decrease seed germination. The additional presence of the biological control strain partly restored the germination level. Introduction of P. putida PCL1760 resulted in significant biological control of TFRR. PCR quantification revealed that the biological control strain decreased the amount of Forl DNA in tomato plant tissue significantly. We conclude that the results of this trial show that P. putida strain PCL1760, which acts through the new mechanism CNN, controls TFRR also under industrial stonewool conditions.     

Effects of cultural treatments on the incidence of, and damage done by, tomato brown root rot

In soils naturally infested with Pyrenochaeta lycopersici, which usually occurs as a grey sterile fungus (GSF), symptoms of brown root rot (BRR) developed sooner and more extensively in the second year of cropping than in the first. The amount of BRR attributable to corkiness increased as plants aged but, at comparable stages of cropping, decreased in the second and third seasons, an effect associated inversely with the severity of early GSF attack. The larger amount of corkiness, 33 compared with 18 %, on two batches of plants in 1965, each with 53 % end-of-season BRR, was also attributed to a less severe early GSF attack on the former than on the latter, growing in soils unsteamed for 1 and 3 years respectively. The incidence of BRR decreased with increasing depth in infested soils but increased on plants grown in plots with partially sterilized topsoil. Partially sterilizing soils at the G.C.R.I. and Fairfield E.H.S. decreased the incidence of BRR and increased crop weights from about the second month of picking, but fruit quality was poorer. Seasonal yields from plants in untreated soil progressively decreased relatively to those from repeatedly steamed plots, from 93 to 65 % in successive years at Fairfield and from 65 to 56 and 43 % at the G.C.R.I. Steaming done in 1963 and 1964 temporarily retarded GSF attack in 1965 with corresponding yield increases. Increasing the amounts of sterilized propagating soil surrounding roots at planting from 0·4 to 1·01 per plant increased yields by c. 0·4 kg/plant, this being a relatively large increase for plants in infested soil, where this treatment significantly delayed the early incidence of BRR near stem bases. Grafting commercially acceptable scions to rootstocks that tolerated colonization by GSF (‘resistant’ rootstocks), temporarily checked growth, delayed picking and decreased fruit quality. Usually grafted plants, irrespective of soil treatment, yielded at least as much fruit as ungrafted plants in steamed soil. In one of five comparisons, soil steaming increased yields of grafted plants. When testing the effects of previous cropping it seemed that populations of GSF increased similarly in soils planted with grafted and ungrafted plants. In addition to GSF attack, roots at Fairfield E.H.S. were often colonized by Colletotrichum coccodes, microsclerotia (= black dots) being more numerous as plants aged. Although significantly more black dot developed on GSF-resistant rootstocks grown in untreated soil than on those grown in steamed soil, the differences were not associated with effects on yield. C. coccodes colonized GSF-susceptible and -resistant roots equally.     

A PCR-RFLP assay for identification and detection of Pythium myriotylum, causal agent of the cocoyam root rot disease

Aim: Development of a PCR‐RFLP assay that could reliably distinguish strains of Pythium myriotylum that are pathogenic to cocoyam from nonpathogens, as well as in planta detection of the pathogen. Methods and Results: Sequences of the internal transcribed spacer regions of nuclear ribosomal DNA (rDNA‐ITS) containing ITS1 and ITS2 of P. myriotylum isolates from cocoyam and other hosts were aligned and a restriction map was generated. rDNA‐ITS alignment report revealed a new single nucleotide polymorphism (SNP; thymine/cytosine) downstream to previously published SNP (guanine/adenine) between isolates of P. myriotylum that are pathogenic to cocoyam and nonpathogenic strains. This new SNP is within the restriction site of the endonuclease AarI. Based on this SNP, a PCR‐RFLP assay was developed for specific detection of P. myriotylum. The PCR amplicons of all isolates of P. myriotylum that infect cocoyam were cleaved by AarI, resulting to two bands (600/400 bp); but those from other hosts showed a single band (1000 bp), confirming the presence and specificity of the AarI restriction site. Also, the assay was effective in in planta detection of the pathogen on infected cocoyam roots without prior isolation of a pure culture. Conclusion: A PCR‐RFLP method was developed that differentiates isolates of P. myriotylum that are pathogenic to cocoyam from nonpathogens as well as from other fungi commonly found in the cocoyam rhizosphere. Significance and Impact of the Study: Early and rapid detection of the pathogen could be of great importance in certifying planting materials as disease‐free, enhancing sustainable management practices and limiting economic losses.     

青稞根腐病防病促生细菌的筛选及其菌剂防效

【背景】由燕麦镰孢(Fusarium avenaceum)和麦根腐平脐蠕孢(Bipolaris sorokiniana)等病原真菌引起的青稞(Hordeum vulgare L. var. nudum Hook. f.)根腐病普遍发生,对藏区农业生产和发展产生了极大威胁。【目的】从青稞健康植株根际土壤中筛选具优良防病促生能力的菌株,通过研究菌株间的互作效应以筛选优良菌剂配伍,制作防效优良的防病促生菌剂,以期为青稞根腐病的防治及青稞增产提供优质的菌剂资源。【方法】采用选择性培养基筛选促生菌株,采用平板对峙法筛选拮抗菌株;采用灌根法和叶面接种法测定菌株致病性;采用乙炔还原法测定固氮能力,钼蓝比色法测定溶磷能力,采用高效液相色谱法测定分泌吲哚乙酸能力;采用16S rRNA基因序列分析鉴定菌种;田间测定菌剂防效,并采用TOPSIS综合分析法进行综合评价。【结果】共分离出具固氮、溶磷能力菌株23株,并从中筛选得到6株拮抗能力良好的菌株,其中5株溶有机磷、3株溶无机磷、3株固氮菌,而且均分泌吲哚乙酸;测定其致病性发现对青稞、燕麦、油菜、豌豆等采样区常见作物均无致病性;经鉴定分别为枯草芽孢杆菌(Ba...     

Fructose-1,6-diphosphate as an in vitro and in vivo anti-alcohol agent in the rat

Fructose-1, 6-diphosphate (FDP) decreases the effect of ethanol on Ca++ entry and inhibits the ethanol-stimulated phosphate efflux in rat heart slices. FDP also inhibits the ethanol-stimulated [36Cl-]-uptake by rat brain microvesicles and affects the isolated GABA-receptor in a way opposite to that of ethanol. The in vivo effects of FDP include a dose-dependent decrease in ethanol-induced gastric ulcers and a decrease in the serum transaminase levels raised by chronic ethanol administration. Other central actions of ethanol such as diuresis, narcosis, dependence and withdrawal symptoms are also counteracted by FDP.     

Hydroxyproline-based DNA mimics provide an efficient gene silencing in vitro and in vivo

To be effective, antisense molecules should be stable in biological fluids, non-toxic, form stable and specific duplexes with target RNAs and readily penetrate through cell membranes without non-specific effects on cell function. We report herein that negatively charged DNA mimics representing chiral analogues of peptide nucleic acids with a constrained trans-4-hydroxy-N-acetylpyrrolidine-2-phosphonate backbone (pHypNAs) meet these criteria. To demonstrate this, we compared silencing potency of these compounds with that of previously evaluated as efficient gene knockdown molecules hetero-oligomers consisting of alternating phosphono-PNA monomers and PNA-like monomers based on trans-4-hydroxy-L-proline (HypNA-pPNAs). Antisense potential of pHypNA mimics was confirmed in a cell-free translation assay with firefly luciferase as well as in a living cell assay with green fluorescent protein. In both cases, the pHypNA antisense oligomers provided a specific knockdown of a target protein production. Confocal microscopy showed that pHypNAs, when transfected into living cells, demonstrated efficient cellular uptake with distribution in the cytosol and nucleus. Also, the high potency of pHypNAs for down-regulation of Ras-like GTPase Ras-dva in Xenopus embryos was demonstrated in comparison with phosphorodiamidate morpholino oligomers. Therefore, our data suggest that pHypNAs are novel antisense agents with potential widespread in vitro and in vivo applications in basic research involving live cells and intact organisms.