Chemical,physical and biological control of carrot seedling diseases

In naturally infested soil containingPythium ultimum, P. acanthicum andPhytophthora megasperma, onlyP. ultimum was associated with root rot and damped-off seedlings. Damping-off was promoted by low soil temperatures and by flooding. Seedling stands were markedly reduced when seed was pre-incubated in soil at 12°C but not at 25°C or 35°C. Dusting carrot seed with metalaxyl significantly increased seedling stands in the field at rates from 1.5–6 g kg⁻¹ seed and in both flooded and unflooded, naturally infested soil at 3.15 g kg⁻¹. In greenhouse experiments using artifically infested soil,P. ultimum andP. paroecandrum caused damping-off of carrot seedlings andRhizoctonia solani reduced root and shoot weights.R. solani caused damping-off in nutrient-enriched soil.P. acanthicum andP. megasperma were not pathogenic to seedlings, although both fungi colonized roots. Soil populations of allPythium spp., particularlyP. ultimum, increased during growth of seedlings and population growth ofP. megasperma was promoted by periodic flooding. Infestation of soil withP. acanthicum did not reduce damping-off of carrot seedlings byP. ultimum orP. paroecandrum, but significantly increased root and shoot weights and decreased root colonization byR. solani P. acanthicum has potential as a biocontrol agent againstR. solani.     

Effect of ultraviolet-induced mutants of Trichoderma harzianum with altered antibiotic production on selected pathogens in vitro

Mutants of Trichoderma harzianum with altered antibiotic production were isolated using ultraviolet light mutagenesis. These included strains whose activity in a Fusarium oxysporum spore germination assay was greater than twice that of the parental strain and one that had no detectable antifungal activity. Characterisation of extracellular metabolites of these strains using thin-layer chromatography and gas-liquid chromatography showed that the strains with high activity produced only elevated levels of a 6-n-pentyl pyrone, the antibiotic produced by the parental strain, but two new antifungal compounds. One of these has been identified as an isonitrile antibiotic. The nature of the interactions of the mutants with Fusarium oxysporum, Rhizoctonia solani, and Pythium ultimum was examined in an in vitro dual-plating assay using two media. High antibiotic production by two T. harzianum strains, BC10 and BC63, did increase inhibition of hyphal growth of R. solani and P. ultimum, but there was no correlation between increased antibiotic production and colonisation ability. In some cases the increased antibiotic levels appeared to impede colonisation of F. oxysporum and R. solani by the mutants. Slow growth rate also affected colonising ability. The types of interactions showed great variability depending on the nature of the T. harzianum isolate and on the test fungus.     

Interaction of the mycoparasite Pythium oligandrum with other Pythium species

Interactions of Pythium oligandrum and four plant‐pathogenic Pythium spp. (P. ultimum, P. vexans, P. graminicola and P. aphanidermatum,) were studied in vitro by (i) video microscopy of hyphal interactions on water agar films, (ii) counting of host and mycoparasite propagules in different regions of opposing colonies on sunflower‐seed extract agar films and (Hi) ability of P. oligandrum to overgrow plates of potato‐dextrose agar previously colonized by Pythium spp. Pythium oligandrum typically coiled round the hyphae of Pythium hosts and penetrated the host hyphae after approximately 50 min from the hyphal coils, causing disruption of host hyphal tips up to 1.2 mm ahead of contact points. The relative growth rates of mycoparasite and host hyphae, timing of penetration and distance (sub‐apical) at which penetration led to host tip disruption were used to assess the potential of mycoparasitism by P. oligandrum to prevent the growth of Pythium hosts. P. aphanidermatum was unique among the ‘host’ Pythium spp. in being largely unaffected by P. oligandrum and in antagonizing the mycoparasite by coiling and penetrating the mycoparasite hyphae. Other host Pythium spp. apparently differed in susceptibility, the most susceptible being P. vexans and P. ultimum, whereas P. graminicola was more resistant. The results are discussed in relation to the role of P. oligandrum as a biocontrol agent, especially for limiting the ability of other Pythium spp. to increase their propagule populations in crop residues.     

Peptide synthetase gene in Trichoderma virens.

Trichoderma virens (synonym, Gliocladium virens), a deuteromycete fungus, suppresses soilborne plant diseases caused by a number of fungi and is used as a biocontrol agent. Several traits that may contribute to the antagonistic interactions of T. virens with disease-causing fungi involve the production of peptide metabolites (e.g., the antibiotic gliotoxin and siderophores used for iron acquisition). We cloned a 5,056-bp partial cDNA encoding a putative peptide synthetase (Psy1) from T. virens using conserved motifs found within the adenylate domain of peptide synthetases. Sequence similarities with conserved motifs of the adenylation domain, acyl transfer, and two condensation domains support identification of the Psy1 gene as a gene that encodes a peptide synthetase. Disruption of the native Psy1 gene through gene replacement was used to identify the function of this gene. Psy1 disruptants produced normal amounts of gliotoxin but grew poorly under low-iron conditions, suggesting that Psy1 plays a role in siderophore production. Psy1 disruptants cannot produce the major T. virens siderophore dimerum acid, a dipetide of acylated N(delta)-hydroxyornithine. Biocontrol activity against damping-off diseases caused by Pythium ultimum and Rhizoctonia solani was not reduced by the Psy1 disruption, suggesting that iron competition through dimerum acid production does not contribute significantly to disease suppression activity under the conditions used.     

Biological control of Pythium damping‐off by seed‐treatment with pseudomonas putida: Relationship with ethanol production by pea and soybean seeds

The relationship between biocontrol activity of Pseudomonas putida strain N1R against Pythium ultimum on pea and soybean seeds and the reduction in ethanol evolution by imbibed seeds was investigated under different treatment conditions, including temperature and numbers of seed‐applied cells of the bacterium. Treatment with strain N1R increased emergence at all temperatures, except for soybean at 12 °C and reduced ethanol concentration in the spermosphere of imbibed seeds at several temperatures. The concentration of bacterial cells in the seed treatment suspension also significantly affected biocontrol efficiency and reduced ethanol production, especially in pea seeds. In contrast, the duration (0–7 h) of submergence of seeds in bacterial suspension had little effect on biocontrol activity of N1R, although submergence of soybean seeds reduced their emergence even in the absence of the pathogen or biocontrol agent. Competition for seed‐derived compounds, including ethanol, is suggested to be one possible mechanism of biocontrol of Pythium by strain N1R, which is not known to produce antifungal antibiotics.     

Timing of gliotoxin biosynthesis in the fungal biological control agentGliocladium virens (Trichoderma virens)

Gliocladium virens is a filamentous fungus formulated for the biological control of damping-off diseases of plants. Part of its antagonistic activity is due to its production of an epidithiodiketopiperazine antibiotic, gliotoxin. A relatively short period of biocontrol activity limits the use of this biocontrol agent in certain applications. This report examines the apparent transient accumulation of gliotoxin, a potential limitation in biocontrol activity.³⁵S pulse labeling of gliotoxin indicated thatG. virens strain G20-4VIB synthesizes gliotoxin only within a short 16-h period during replicative growth. An apparent lack of gliotoxin production in later growth phases was due to the cessation of synthesis rather than an increase in gliotoxin catabolism. Media-trnafer experiments indicated that cessation of gliotoxin synthesis could not be explained by gliotoxin feedback inhibition, a diffusible inhibitor, or changing the nutritional status of the medium over a 2-h response time. These results demonstrate that the regulation of gliotoxin biosynthesis is a major determinant in the kinetics of gliotoxin appearance and focuses the need for further study on the regulation of gene expression.     

毒死蜱降解木霉菌对几种重要植物病原真菌的生防活性

木霉菌既是广泛应用的防治植物病害的生防菌,又是一类很有应用潜力的环境污染修复菌。针对分离筛选出的6株高效降解毒死蜱的木霉菌株,进行了土传植物真菌病害的生防活性试验。结果表明,在对峙培养条件下,供试木霉菌株对几种病原真菌均具有较为显著的抑制率,发酵滤液对多数病原真菌具有明显的抑菌作用。所有供试木霉菌株能在立枯丝核菌、灰霉、终极腐霉菌落上着生,并逐渐覆盖全部菌落;但不能在茄腐镰孢菌、尖孢镰孢菌、大丽轮枝菌上生长。真菌重寄生现象观察结果表明,供试木霉菌仅对立枯丝核菌具有明显的重寄生现象。研究结果表明,筛选出的高效降解毒死蜱的木霉菌菌株可对多种土传植物病原真菌具有良好的生防潜力。     

Influence of Culture Conditions on Growth and Survival of Conidia of Trichoderma spp. Coated on Seeds

Five Trichoderma strains were grown on rice, on vermiculite plus potato-dextrose broth (PDB), on potato-dextrose agar (PDA) or in liquid cultures supplemented with glycerol, KCl or polyethylene glycol (PEG) at -1 MPa or - 2 MPa. Conidia were coated on seeds through a methyl cellulose coating or through an industrial film-coating process. The conidial yield decreased with glycerol, KCl or PEG compared with PDB alone. The percentage viability was from 23 to 44% after methyl cellulose coating, regardless of the culture conditions for conidial production. In general, the industrial coating resulted in lower numbers of living conidia. The viability during storage was enhanced when vermiculite, rice or PDA were used as substrates for fungal growth. Nevertheless, temperature of storage was found to be more critical to spore survival than the substrate used for spore production; conidial viability on seeds did not exceed 4 months at 15 C. Solid and liquid cultures produced conidia able to control R. solani and P. ultimum when applied to seeds through industrial film coating. The level of disease suppression varied with the number of viable conidia/seed and with the culture medium used for conidial production. The three main conditions for further industrial application-high yields, longevity and biocontrol effectiveness-might be optimized by selecting the appropriate medium (liquid or solid), water potential and solutes used.     

Induction of cytoplasmic leakage from Rhizoctonia solani hyphae by Gliocladium virens and partial characterization of a leakage factor

The biocontrol fungus Gliocladium virens (Gl‐21)was grown on various solid and liquid substrates. Aqueous extracts of wheat bran and peanut hull meal (PHM)as well as spent glucose tartrate broth (GTB), Czapek‐Dox broth (CDB) and potato dextrose broth (PDB) upon which Gl‐21 was grown caused leakage of carbohydrates and electrolytes from hyphae of the soilborne plant pathogen Rhizoctonia solani. In addition, the mycelial weight of R. solani was reduced. Most of the leakage factor was formed in the substrates within six days of incubation. Acidification of the bran and the PHM media before inoculation stimulated production of the leakage factor by G. virens. Size‐fractionation experiments indicated that a combination of factors produced by G. virens induced leakage from R. solani. Although the < 1 k Da fraction from the water‐extracted bran culture contained significant leakage activity, it was less than in the non‐fractionated bran culture. The > 1 k Da fraction from bran culture did not induce leakage activity. When the < 1 k Da and the > 1 k Da fractions were recombined, leakage activity was restored to a level similar to that of the non‐fractionated preparation. Gliotoxin was detected in culture filtrates from G. virens grown on bran and PHM media. Gliotoxin preparations induced leakage of carbohydrates and electrolytes from R. solani and caused a concomitant reduction in mycelial weight, which suggests that it is a leakage factor.     

亚麻立枯病拮抗菌的筛选、生防效果及发酵条件优化

【目的】从健康亚麻植株的根际土壤中筛选对亚麻立枯病菌具有较强抑菌作用的拮抗菌,优化其产生抑菌活性物质的发酵条件,为其生防利用奠定基础。【方法】采用稀释平板涂布法和对峙培养法进行拮抗菌的筛选;根据菌株形态学特征、生理生化特性以及16S r RNA基因序列分析对其进行鉴定;利用温室抗病实验确定其生防效果;通过单因素实验和均匀设计实验优化其发酵条件。【结果】分离筛选到一株对亚麻立枯病菌具有显著拮抗作用的细菌HXP-5,且其对另外7种植物病菌真菌均有拮抗作用;鉴定菌株HXP-5为枯草芽孢杆菌;温室抗病实验结果表明其生防效果可达71.22%;其产生抑菌活性物质的最佳发酵条件为:葡萄糖为2.3%,胰蛋白胨+酵母粉(3:1)为0.25%,Na Cl为0.18%,发酵时间为72 h,发酵温度为27°C,转速为210 r/min,250 m L摇瓶装液100 m L,接种量为1.7%。【结论】经鉴定,对亚麻立枯病病菌具拮抗作用的菌株HXP-5为枯草芽孢杆菌,且对亚麻立枯病具有较强的防治效果,发酵条件进行优化后其对亚麻立枯病病原菌显示出更强的拮抗作用。     

Biocontrol of fungal root rot diseases of crop plants by the use of rhizobia and bradyrhizobia

Twenty-oneRhizobium andBradyrhizobium strains were testedin vitro against the mycelial growth of three pathogenic fungi on solid and liquid media. All tested rhizobia and bradyrhizobia significantly suppressed the growth of the three soil-borne root-infecting fungi (Fusarium solani, Macrophominia phasolina andRhizoctonia solani) either in the absence or presence of iron. This indicates that the siderophore played a minor role in the biocontrol potential ofRhizobium andBradyrhizobium against pathogenic fungi. Pot experiments revealed that the numbers of propagules causing disease after 4 weeks of planting varied with species and host plant. The three most activeRhizobium andBradyrhizobium strains (R. leguminosarum bv.phaseoli TAL 182,B. japonicum TAL 377 andBradyrhizobium sp. (lupin) WPBS 3211 D) tested under greenhouse conditions for their ability to protect one leguminous (soybean) and two non-leguminous (sunflower and okra) seedlings from root rot caused byFusarium solani, Macrophominia phaseolina andRhizoctonia solani provided significant suppression of disease severity compared with nonbacterized control in both leguminous and non-leguminous seedlings.Bradyrhizobium sp. (lupin) WPBS 3211 D provided the lowest degree of resistance against all the tested pathogens with all host plants. *** DIRECT SUPPORT *** A00EN058 00013     

Phytoplasma Transmission by Heterologous Grafting Influences Viability of the Scion and Results in Early Symptom Development in Periwinkle Rootstock

The stolbur phytoplasma ‘Candidatus Phytoplasma solani’ is responsible for the grapevine disease ‘bois noir’ affecting a number of wine‐growing areas in Europe. Transmission of stolbur phytoplasma to different laboratory hosts can be difficult due to the requirement of transmitting insect vectors or parasite plants. Here, heterologous grafting was used as an alternative technique for transmission of common and strongly symptomatic stolbur genotypes CPsM4_At1 and CPsM4_At6 of ‘Ca. P. solani’ to experimental host plants such as Catharanthus roseus and tomato making phytoplasma strains more accessible for molecular and experimental investigations in different plant species. Transmission was confirmed by quantitative PCR, microscopy and nested PCR followed by marker gene sequencing. In our study, the transmission of different genotypes of ‘Ca. P. solani’ resulted in distinguishable symptom development in the laboratory host C. roseus. Symptom development in grafted rootstock was observed three to 7 weeks after heterologous grafting. Survival of the graft unit was influenced by the presence of ‘Ca. P. solani’ in the scions and was clearly reduced in phytoplasma free scion – rootstock combinations.     

Relationship of in vitro and in planta screening: improving the selection process for biological control agents against Fusarium root rot in row crops

Fusarium root rot in row crops is typically managed by cultural practices and fungicide seed treatments. Biological control using microbial agents is another option but needs further development for improved disease management. Screening to identify biocontrol agents are crucial. However, relationships among the steps and how to improve the screening process are unresolved questions. Strains of Burkholderia (4), Bacillus (5) and Trichoderma (26) were studied in vitro against six Fusarium pathogens. All the bacteria and five selected Trichoderma strains were tested in planta in the greenhouse against diseases of Fusarium graminearum and Fusarium oxysporum. Burkholderia ambifaria C628, Bacillus simplex R180, and all Trichoderma isolates showed high reduction in disease levels in corn, soybean and wheat, ranging from 16 to 63%. Responses of the biocontrol agents during in vitro and in planta screening did not always correlate. In vitro and in planta tests should be considered independently in selecting biocontrol candidates.     

Molecular identification of diverse ‘Candidatus Phytoplasma’ species associated with grapevine decline in Iran

Grapevine (Vitis vinifera) is one of the most important fruits in Iran where the provinces of Qazvin, Lorestan and Markazi are main producers. During 2013–2015, vineyards located in these provinces were surveyed to verify the presence of phytoplasma. The sample collection was based on symptomatology including decline, leaf yellowing and shortening of internodes. Total DNA was extracted from symptomatic and symptomless grapevine samples and used in nested‐polymerase chain reaction (PCR) assays with phytoplasma ribosomal primers (P1/Tint followed by R16F2n/R2, R16mF1/mR1, R16(I)F1/R1 or 6R758f/16R1232r). Nested‐PCR products were obtained only for symptomatic samples while samples from symptomless plants yielded no PCR products. Restriction fragment length polymorphism (RFLP) analyses with Tru1I, TaqI and Tsp509I and direct sequencing of amplicons followed by phylogenetic analyses indicated the presence of ‘Candidatus Phytoplasma fraxini’, ‘Ca. P. aurantifolia’, ‘Ca. P. solani’ and ‘Ca. P. phoenicium’‐related strains. In Marzaki province, there ‘Ca. P. aurantifolia’ strains were mainly detected, while in the other two provinces, all the four ‘Candidatus species’ were identified with the prevalence of ‘Ca. P. solani’‐related strains. In both provinces in one case, mixed phytoplasma infection was also detected by RFLP analyses. The presence of different phytoplasmas in positive samples indicates great phytosanitary significance due to grapevine economic importance for country. Grapevine phytoplasma infection represents a threat for other crops suggesting grapevine as alternative host species for the phytoplasmas already reported in Iran, while the ‘Ca. P. fraxini’ is for the first time identified in Iran.     

Potential of an antagonistic bacterium isolate obtained fromLentinus lepideus basidiospores as a biocontrol agent

TheBurkholderia sp. isolate 87-11 obtained from basidiospores ofLentinus lepideus was antagonistic against severalPythium andRhizoctonia isolates. The bacterium was tested against soilborne diseases of five plants caused byP. aphanidermatum andR. solani by soil and seed application, and its potential as a biocontrol agent is discussed.     

Characteristics of aSerratia plymuthica isolate from plant rhizospheres

An isolate (G15) of a bacterium, frequently isolated from roots of various plant species, was identified asSerratia plymuthica. At low temperature (viz. 2–8°C), the studied isolated readily produced a red pigment which proved useful in recognizing the bacteria on reisolation. In laboratory tests it exhibited strong antagonism againstBotrytis cinerea andGerlachia nivalis and moderate antagonism againstRhizoctonia solani, Fusarium culmorum andPythium sp. The bacterium significantly increased growth of lettuce plants when applied to the roots under non-sterile conditions in greenhouse tests. Various strains ofSerratia plymuthica are supposed to be common as rhizosphere bacteria under Swedish conditions.     

Isolation and characterization of endophytic Streptomyces strains from surface-sterilized tomato (Lycopersicon esculentum) roots

AIMS: To isolate endophytic Streptomyces strains from tomato and examine their antimicrobial activity. METHODS: Endophytic Streptomyces strains were isolated using surface-sterilization methods and identified by morphological characteristics. Antimicrobial activities were measured by the agar plate sensitivity method. Antifungal activity in vivo was measured by seedling mortality in infested soils. RESULTS: Twenty-one per cent of endophytic streptomycete isolates produced antibacterial metabolites and 41% produced antifungal metabolites in S medium. Sixty-five per cent of the most frequently isolated strains inhibited the growth of Rhizoctonia solani by the antibiosis assay but only 32% produced metabolites active against R. solani in S medium. Growth promotion and enhanced disease resistance of seedlings inoculated with Streptomyces sp. strain S30 were observed in tomato but not in cucumber seedlings. CONCLUSIONS: Endophytic Streptomyces spp. strains were successfully isolated using stringent methods and strain S30 promoted growth and enhanced resistance to R. solani in tomato seedlings. SIGNIFICANCE AND IMPACT OF THE STUDY: Endophytic streptomycetes showing antifungal activity in vitro and in vivo may indicate the potential for their use as biocontrol agents particularly of R. solani disease of tomato seedlings.     

Osmotic effects on radial growth rate and specific growth rate of three soil fungi

The radial growth rate on osmotically adjusted agar medium and the relative specific growth rate in osmotically adjusted liquid medium were determined for Rhizoctonia solani, Pythium ultimum, and Verticillium dahliae. On basal medium, an isolate of P. ultimum and R. solani had similar radial growth rates of 0.52 and 0.47 mm/h, respectively, whereas V. dahliae grew at a rate of 0.08 mm/h. Radial growth rate was reduced 50% at osmotic potentials of -16, -27, and -32 bars for P. ultimum, R. solani, and V. dahliae, respectively. No growth occurred at -32 bars for P. ultimum, -56.2 bars for R. solani, and -100 bars for V. dahlia. Specific growth rates in liquid culture were 0.011 h-1 for P. ultimum, 0.008 h-1 for V. dahliae, and 0.026 h-1 for R. solani. Ratios of radial growth rate (Kr) to specific growth rate (alphas) were computed for each fungus growing at different osmotic potentials. There was not a constant relationship between Kr on agar and alphas in liquid medium, e.g., Kr/alphas ratios varied from 8-41% from a mean ratio for a particular species. The results indicated that radial growth rate on osmotic agar was not useful as a measure of relative specific growth rate of a fungus in osmotically adjusted liquid medium.     

Biocontrol of Pythium in the pea rhizosphere by antifungal metabolite producing and non-producing Pseudomonas strains

AIMS: Four well-described strains of Pseudomonas fluorescens were assessed for their effect on pea growth and their antagonistic activity against large Pythium ultimum inocula. Methods and RESULTS: The effect of Pseudomonas strains on the indigenous soil microflora, soil enzyme activities and plant growth in the presence and absence of Pythium was assessed. Pythium inoculation reduced the shoot and root weights, root length, and the number of lateral roots. The effect of Pythium was reduced by the Pseudomonas strains. Strains F113, SBW25 and CHAO increased shoot weights (by 20%, 22% and 35%, respectively); strains Q2-87, SBW25 and CHAO increased root weights (14%, 14% and 52%). Strains SBW25 and CHAO increased root lengths (19% and 69%) and increased the number of lateral roots (14% and 29%). All the Pseudomonas strains reduced the number of lesions and the root and soil Pythium populations, while SBW25 and CHAO increased the number of lateral roots. Pythium inoculation increased root and soil microbial populations but the magnitude of this effect was Pseudomonas strain-specific. Pythium increased the activity of C, N and P cycle enzymes, while the Pseudomonas strains reduced this effect, indicating reduced plant damage. CONCLUSION: Strains SBW25 and CHAO had the greatest beneficial characteristics, as these strains produced the greatest reductions in the side effects of Pythium infection (microbial populations and enzyme activities) and resulted in significantly improved plant growth. Strain SBW25 does not produce antifungal metabolites, and its biocontrol activity was related to a greater colonization ability in the rhizosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first critical comparison of such important strains of Ps. fluorescens showing disease biocontrol potential.     

Enhanced biocontrol activity of Trichoderma virens transformants constitutively coexpressing β-1,3- and β-1,6-glucanase genes

Evidence for the role of chitinases, proteases and β-1,3- and β-1,6-glucanases in mycoparasitism by Trichoderma species has been well documented. Moreover, constitutive over-expression of genes encoding individual cell-wall-degrading enzymes (CWDEs) has been shown to improve the potential of biological agents. In this study, we generated transformants of T. virens in which β-1,3- and β-1,6-glucanase genes, TvBgn2 and TvBgn3 , respectively, were constitutively coexpressed in the same genetic T. virens Gv29.8 wild-type background. The double over-expression transformants (dOEs) grow and sporulate slower than the wild-type (WT). However, the reduction in growth did not seem to affect their mycoparasitic and biocontrol capabilities, as dOEs displayed much higher levels of total β-1,3- and β-1,6-glucanase activity than the WT. This higher enzymatic activity of dOEs positively correlated with observed in vitro inhibition of Pythium ultimum and Rhizoctonia solani mycelia, and with enhanced bioprotection of cotton seedlings against P. ultimum , R. solani and Rhizopus oryzae . Besides effective biocontrol of all pathogens at an original inoculum level, the performance of dOEs was highly enhanced (up to 312% of WT performance) when pathogen pressure was greater (i.e. concentration of inoculum was higher or pathogens applied in combination). These results demonstrate that the strategy of introducing multiple lytic enzyme-encoding genes through transformation of a given biocontrol strain can be successfully used to achieve better biocontrol.