Ultrastructure and germinability of Verticillium dahliae microsclerotia parasitized by Talaromyces flavus on agar medium and in treated soil

The interactions between microsclerotia (ms) of the fungal plant pathogen Verticillium dahliae and the mycoparasite Talaromyces flavus were followed in soil and on agar medium. Germinability of ms, which had been incubated for 14 days in soil treated with 0.5% of a T. flavus ‐ wheat bran preparation, decreased from 84% to 17%, as compared with 81% and 74% in untreated soil and in soil treated with a sterilized biocontrol preparation respectively. Germinability of ms which had been buried in treated soil for 4 days decreased to 70%, all ms being parasitized by T. flavus. Upon transfer of the ms to untreated soil for 10 more days, germinability decreased further to 20%, indicating that T. flavus continued to parasitize sclerotia in the untreated soil. Scanning electron micrographs showed heavy fungal colonization and typical T. flavus conidia on the surface of the ms buried in the treated soil, but not in control soils. Transmission electron micrographs of ms incubated with T. flavus on agar revealed parsitism involving invasion of some host cells by means of small penetration pegs; the host cell walls were mainly lysed at their site of contact with the parasite hyphal tips. Further colonization of the ms cells occurred simultaneously with the degradation of the invaded host cell contents, rather than the cell walls. Mycoparasitism of V. dahliae ms by T. flavus hyphae may be involved in the biological control of verticillium wilt disease.     

Expression of the Talaromyces flavus glucose oxidase gene in cotton and tobacco reduces fungal infection,but is also phytotoxic

Glucose oxidase secreted by the fungus Talaromyces flavus generates, in the presence of glucose, hydrogen peroxide that is toxic to phytopathogenic fungi responsible for economically important diseases in many crops. A glucose oxidase gene from T. flavus, was modified with a carrot extensin signal peptide and fused to either a constitutive or root-specific plant promoter. T1 tobacco plants expressing the enzyme constitutively were protected against infection by the seedling pathogen Rhizoctonia solani. Constitutive expression in tobacco was associated with reduced root growth, and slow germination on culture medium, and with reduced seed set in glasshouse conditions. Several independent transformed cotton plants with a root-specific construct expressed high glucose oxidase activity in the roots, excluding the root tip. Selected T3 homozygous lines showed some protection against the root pathogen, Verticillium dahliae, but not against Fusarium oxysporum. High levels of glucose oxidase expression in cotton roots were associated with reduced height, seed set and seedling germination and reduced lateral root formation. If this gene is to be of value for crop protection against pathogens it will require precise control of its expression to remove the deleterious phenotypes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Functional Analysis of Autophagy Genes via Agrobacterium-Mediated Transformation in the Vascular Wilt Fungus Verticillium dahliae

Autophagy is a widely conserved intracellular process for degradation and recycling of proteins,organelles and cytoplasm in eukaryotic organisms and is now emerging as an important process in foliar in...     

Relationship between soil densities of Aspergillus species and colonization of wounded peanut seeds

Soil is a reservoir for Aspergillus flavus and A. parasiticus, fungi that commonly colonize peanut seeds and produce carcinogenic aflatoxins. Densities of these fungi in soil vary greatly among fields and may influence the severity of peanut infection. This study examined the relationship between soil density of Aspergillus species and the incidence of peanut seed colonization under laboratory conditions. Viable peanut seeds were wounded and inoculated with 20 soils differing in composition and density of Aspergillus species and were then incubated for 14 days at 37 degrees C (seed water activity = 0.92). The effect of soil density of individual section Flavi species (A. flavus strains L and S, A. parasiticus, A. caelatus, and A. tamarii), section Nigri, and A. terreus on the incidence of seed colonization was best expressed as a function of exponential rise to maximum. Exponential curves often rose to maximum percentages of seed colonization by section Flavi species that were well below 100% despite high species densities in some soils. Competition primarily among section Flavi species may explain the reduced incidences of seed colonization. An average of two or fewer propagules of each Aspergillus species in the soil at the wound site was required for colonization of 20% of peanut seeds. Other fungal species were capable of invading peanut seeds only when soil densities of sections Flavi and Nigri species were low.     

Colonization of rye green manure and peanut fruit debris by Aspergillus falvus and Aspergillus niger group in field soils.

Aspergillus flavus and Aspergillus niger group colonization of deep-plowed, decomposing rye green manure cover crops in peanut field soils was studied in four fields during 1972 and 1973; colonization of decomposing peanut fruits was studied in 1972 in two fields. A. flavus colonization of rye and peanut fruits was greater in soils of heavy texture, and an A. flavus population as high as 165 propagules per g of soil was observed in soil adjacent to rye, whereas A. flavus populations in soils not associated with rye were 18 propagules per g of soil or lower. Highest A. flavus populations in soil adjacent to decomposing peanut fruits were usually comparable to populations associated with rye. Little decomposing rye or peanut fruit colonization was generally observed by the A. flavus competitor, A. niger group. A. flavus may maintain or increase its inoculum potential by colonization of these and other moribund plant tissues.     

Effect of Initial Nematode Population Density on the Interaction of Pratylenchus penetrans and Verticillium dahliae on 'Russet Burbank' Potato

Four similar growth chamber experiments were conducted to test the hypothesis that the initial population density (Pi) of Pratylenchus penetrans influences the severity of interactive effects of P. penetrans and Verticillium dahliae on shoot growth, photosynthesis, and tuber yield of Russet Burbank potato. In each experiment, three population densities of P. penetrans with and without concomitant inoculation with V. dahliae were compared with nematode-free controls. The three specific Pi of JR penetrans tested varied from experiment to experiment but fell in the ranges 0.8-2.5, 1.8-3.9, 2.1-8.8, and 7.5-32.4 nematodes/cm³ soil. Inoculum of V. dahliaewas mixed into soil, and the assayed density was 5.4 propagules/gram dry soil. Plants were grown 60 to 80 days in a controlled environment. Plant growth parameters in two experiments indicated significant interactions between P. penetrans and V. dahliae. In the absence of V. dahliae, P. penetrans did not reduce plant growth and tuber yield below that of the nematode-free control or did so only at the highest one or two population densities tested. In the presence of K dahliae, the lowest population density significantly reduced shoot weight and photosynthesis in three and four experiments, respectively. Higher densities had no additional effect on shoot weight and caused additional reductions in photosynthesis in only one experiment. Population densities of 0.8 and 7.5 nematodes/cm³ soil reduced tuber yield by 51% and 45%, whereas higher densities had no effect or a 15% additional effect, respectively. These data indicate that interactive effects between P. penetrans and V. dahliae on Russet Burbank potato are manifested at P. penetrans population densities less than 1 nematode/cm³ soil and that the nematode population density must be substantially higher before additional effects are apparent.     

In vitro analysis of the role of glucose oxidase from Talaromyces flavus in biocontrol of the plant pathogen Verticillium dahliae.

Culture filtrates from Talaromyces flavus grown on glucose contained high levels of glucose oxidase activity, while culture filtrates from T. flavus grown on xylan contained negligible glucose oxidase activity. Culture filtrates from T-flavus grown on both media contained complex protein profiles. However, only culture filtrates from T. flavus grown on glucose inhibited germination of microsclerotia of Verticillium dahliae in in vitro inhibition assays. A polyclonal antiserum preparation, pABGO-1, raised against purified glucose oxidase from T. flavus was highly specific for glucose oxidase. Only one protein band in culture filtrates (from glucose medium), migrating at 71 kDa, was detected in Western blots (immunoblots) with this antiserum. This band comigrated with purified glucose oxidase. No bands were detected in culture filtrates from the xylan medium. Glucose oxidase was removed via immunoprecipitation from culture filtrates of T. flavus grown in glucose medium, resulting in filtrates which no longer inhibited in vitro microsclerotial germination. When glucose oxidase-depleted filtrates were amended with purified glucose oxidase from T. flavus, the ability to kill microsclerotia in vitro was restored to original levels. We conclude that glucose oxidase is the only protein in culture filtrates of T. flavus responsible for inhibition of germination of microsclerotia of V. dahliae.     

Processed Manure as Carrier to Introduce Trichoderma harzianum: Population Dynamics and Biocontrol Effect on Rhizoctonia solani

Manure pellets produced from processed swine faeces can be used as carrier material for the biocontrol fungus Trichoderma harzianum. The antagonist can grow and sporulate on the processed manure powder as the sole source of carbon and nutrients. The incorporation of conidia in pellets of the processed manure was shown to be feasible on a laboratory scale. Survival of the fungus in the pellets during storage was satisfactory. The population dynamics of T. harzianum were studied using a benomyl-resistance marker after introduction of conidia into soil. The antagonist could colonize and spread through a number of non-sterile soils and was able to establish a stable population over a period exceeding 125 days. Under sterile conditions, the propagation of T. harzianum in soil was much greater than under non-sterile conditions. The incorporation of antagonist conidia in pellets was found to be essential for the successful colonization of non-sterile soil. In growth chamber experiments, application of T. harzianum via processed manure pellets reduced damping-off of sugar beet seedlings caused by Rhizoctonia solani in artificially and naturally infested soil. In artificially infested soil, T. harzianum reduced the population of R. solani and protected beet seedlings from damping-off 3 weeks after introduction. The application of T. harzianum to naturally infested soil increased the number of healthy beet seedlings more than two-fold.     

Suppression of Rhizoctonia solani diseases of sugar beet by antagonistic and plant growth-promoting yeasts

AIMS: Isolates of Candida valida, Rhodotorula glutinis and Trichosporon asahii from the rhizosphere of sugar beet in Egypt were examined for their ability to colonize roots, to promote plant growth and to protect sugar beet from Rhizoctonia solani AG-2-2 diseases, under glasshouse conditions. METHODS AND RESULTS: Root colonization abilities of the three yeast species were tested using the root colonization plate assay and the sand-tube method. In the root colonization plate assay, C. valida and T. asahii colonized 95% of roots after 6 days, whilst Rhod. glutinis colonized 90% of roots after 8 days. Root-colonization abilities of the three yeast species tested by the sand-tube method showed that roots and soils attached to roots of sugar beet seedlings were colonized to different degrees. Population densities showed that the three yeast species were found at all depths of the rhizosphere soil adhering to taproots up to 10 cm, but population densities were significantly (P < 0.05) greater in the first 4 cm of the root system compared with other root depths. The three yeast species, applied individually or in combination, significantly (P < 0.05) promoted plant growth and reduced damping off, crown and root rots of sugar beet in glasshouse trials. The combination of the three yeasts (which were not inhibitory to each other) resulted in significantly (P < 0.05) better biocontrol of diseases and plant growth promotion than plants exposed to individual species. CONCLUSIONS: Isolates of C. valida, Rhod. glutinis and T. asahii were capable of colonizing sugar beet roots, promoting growth of sugar beet and protecting the seedlings and mature plants from R. solani diseases. This is the first successful attempt to use yeasts as biocontrol agents against R. solani which causes root diseases. SIGNIFICANCE AND IMPACT OF THE STUDY: Yeasts were shown to provide significant protection to sugar beet roots against R. solani, a serious soil-borne root pathogen. Yeasts also have the potential to be used as biological fertilizers.     

Root-colonizing Fungi from Oilseed Rape and their Inhibition of Verticillium dahliae

Root-colonizing fungi obtained from thoroughly washed root segments of symptomless young oilseed rape plants growing in field soil were examined for their inhibitory effect on the wilt fungus, Verticillium dahliae Kleb. The isolated fungi that were possible to identify belonged to the different genera Trichoderma , Gliocladium , Mortierella , Fusarium and Alternaria . Oilseed rape seedlings from seeds exposed to both sporulating and nonsporulating fungal isolates were shown to be colonized by their hyphae in gnotobiotic conditions and the emergence/root growth was significantly affected in their presence. Inoculation with a few isolates not only covered the root surface but also seemed to extensively colonize the inner root cortical tissues partly by producing characteristic structures in the roots. All of these suppressed the pathogen in in vitro assays but the mode of suppression in most cases did not seem to be due to production of antifungal substances. In nonsterile field soil, several of the isolates conferred protection against V. dahliae by delaying symptom development. None of the isolates was shown to increase wilt symptoms. In addition to protection, two isolates, yet to be identified, also showed increased shoot dry weight compared with that of healthy controls. A majority of the isolates showed proteolytic activity whereas only three produced chitinolytic enzymes.
Results for the isolates obtained by the method selected for this study could be considered as a starting point for evaluating antagonists capable of both competing for root colonization and preventing root infection, with the final aim being to achieve successful biological control of the wilt fungus.     

Overexpression of GhPFN2 enhances protection against Verticillium dahliae invasion in cotton

Growing evidence indicates that actin cytoskeleton is involved in plant innate immune responses,but the functional mechanism remains largely unknown.Here,we investigated the behavior of a cotton profilin gene(GhPFN2) in response to Verticillium dahliae invasion,and evaluated its contribution to plant defense against this soil-borne fungal pathogen.GhPFN2 expression was up-regulated when cotton root was inoculated with V.dahliae,and the actin architecture was reorganized in the infected root cells,with a clear increase in the density of filamentous actin and the extent of actin bundling.Compared to the wild type,GhPFN2-overexpressing cotton plants showed enhanced protection against V.dahliae infection and the actin cytoskeleton organization in root epidermal cells was clearly altered,which phenocopied that of the wild-type(WT) root cells challenged with V.dahliae.These results provide a solid line of evidence showing that actin cytoskeleton reorganization involving GhPFN2 is important for defense against V.dahliae infection.     

Survival ofalginate-ecapsulated Pseudomonas fluorescens cells in soil

Alginate-encapsulated and unencapsulated cells of Pseudomonas fluorescens Rsf were introduced into soil microcosms with and without wheat plants to evaluate bacterial survival and colonization of the rhizoplane and rhizosphere. Encapsualtion of cells in alginate amended with skim milk or with skim milk plus bentonite clay significantly enchanced long-term survival of the cells. There was a negligible effect on long-term bacterial survival when cells were encapsulated in alginate amended with TY medium or soil extract, as compared to water. Drying of beads resulted in a significant reduction in bacterial viability. After addition to soil, cells in dried beads increased in numbers and exhibited stable population densities, whereas cells added in moist beads showed stable dynamics at a higher level. Cells encapsulated in dried beads or fresh beads survived better than unencapsulated cells added to soil. Both cells in moist and dried alginate beads also survide a dry/wet cycle in soil, whereas unencapsulated cells were sensitive to these moisture fluctuations. Shortly after inoculation and 63 days after this, cells from moist beads colonized wheat roots at significantly higher levels than unencapsulated cells, whereas cells in dried beads did so at levels similat to unencapsulated cells. Cells in beads initially placed at different distance from developing root mat were able to move towards and colonize the rhizosphere, at levels of roughly 10⁴ to 10⁶ colony-forming units fo P. fluorescens R2f per gram of dry soil. Correspondence to: J. T. Trevors or J. D. van Elsas     

Role of Zinc in Rhizobacteria-Mediated Suppression of Root-Infecting Fungi and Root-Knot Nematode

Understanding the environmental factors that influence the rhizosphere and inner root colonization of the disease‐suppressive strains of fluorescent pseudomonads is an essential step towards improving the level and reliability of their biocontrol activity. Soil amendment with Zn at 0.8 or 1.6 mg/kg of soil alone or in combination with Pseudomonas aeruginosa IE‐6S⁺significantly reduced nematode penetration in tomato roots. Zn applied alone did not reduce root infection caused by Macrophomina phaseolina or Fusarium solani but did reduce when used in combination with IE‐6S⁺. Soil amendment with Zn at 0.8 or 1.6 mg/kg of soil alone or in conjunction with IE‐6S⁺ markedly suppressed Rhizoctonia solani infection. Plant height, fresh weight of shoot and protein contents of the leaves substantially improved when used with Zn, however, plants growing in the soil treated with 1.6 mg/kg of Zn in the absence of IE‐6S⁺ not only reduced plant growth but also showed necrotic symptoms on the leaves. Zn application in the soil decreased populations of IE‐6S⁺ both in the rhizosphere and root. A positive correlation between bacterial rhizosphere and inner root colonization was also observed. With an increase in nematode densities in the soil, nematode penetration and subsequent galling due to Meloidogyne javanica increased. Regardless of the nematode densities, Zn applied alone or in combination with IE‐6S⁺ caused marked suppression of M. javanica. At all the population densities of M. javanica, Zn enhanced the efficacy of IE‐6S⁺ to reduce nematode invasion and subsequent gall development. IE‐6S⁺ caused significant suppression of soil‐borne root‐infecting fungi both in Zn‐sufficient and Zn‐deficient soil although this suppressive effect accentuated in Zn‐sufficient soils. In the absence of IE‐6S⁺ and/or Zn, increased nematode densities in the soil significantly reduced plant height, fresh weight of shoot and protein contents of the shoots. With an increase in nematode densities, populations of IE‐6S⁺ in the rhizosphere and root increased regardless of the Zn application. However, Zn‐deficient soils supported larger populations of IE‐6S⁺ compared with those of Zn‐sufficient soils.     

Effect of Tillage and Water Management on the Population Behaviour of Root-Knot Nematodes Meloidogyne Triticoryzae in Rice Crop

The effects of puddling and water regimes on hydraulic conductivity (cm/day) of soil and on bulk density (mg/m 3 ) during rice culture, soil physical characteristics of the experimental field population densities of plant-parasitic nematodes have been studied. Puddling reduced the bulk density of soil and decreased the hydraulic conductivity in the upper layers but not in the deeper layers of soil aeration was reduced due to high moisture levels retained in the puddled soil. Population density of M. triticoryzae declined in puddled soil. The invasion of the roots by the second-generation infective juveniles was reduced. The population density of the root-knot nematodes was higher in the non-puddled soil especially in unsubmerged condition compared to puddled and submerged soil. However, if the seedlings were already infected before transplanting and submergence, the nematode could survive well and reproduce within the aerenchyma of the root.     

Factors influencing colonization of cucumber roots by Clonostachys rosea f. catenulata,a biological disease control agent

The biocontrol fungus Clonostachys rosea f. catenulata (Gliocladium catenulatum) strain J1446, commercially available as Prestop® (Verdera Oy, Finland), is an effective antagonist against several root and foliar greenhouse pathogens. The biocontrol agent forms dense networks of hyphae on plant roots, grows internally in root epidermal cells, and produces hydrolytic enzymes, all of which lead to a reduction in pathogen propagules. An understanding of the environmental and host factors that influence root colonization by C. rosea f. catenulata is important to maximize disease control efficacy. Cucumber roots grown in nutrient solution in containers were inoculated with conidia of a GUS-transformed strain of C. rosea f. catenulata. Population levels associated with roots over time were assessed by colony-plate counts, GUS staining and enzymatic assays to determine GUS activity. Variables such as pH, temperature and growing medium were major factors that influenced population levels, while cucumber cultivar, addition of nutrients, and wounding of roots did not appear to significantly affect colonization. Population density of C. rosea f. catenulata on roots was highest when the nutrient solution was maintained at pH 5, 6, or 7, and at temperatures of 18–22°C. Lowest colonization levels were observed on roots of plants grown in potting mix or in field soil. Measurement of GUS activity provided a slightly more accurate assessment of root colonization levels compared to colony-plate counts. These results illustrate the optimal environmental conditions which can ensure maximum root colonization by C. rosea f. catenulata and enhance disease control by the biocontrol agent.     

Relationships among soil properties, plant nutrition and arbuscular mycorrhizal fungi–plant symbioses in a temperate grassland along hydrologic, saline and sodic gradients

Temporal variations in the relationships among plant nutrient concentrations, soil properties and arbuscular-mycorrhizal (AM) fungal dynamics were studied along a topographic and saline gradient in a temperate grassland soil. Soil and plant ( Lotus tenuis , Paspalum vaginatum , Stenotaphrum secundatum ) samples were collected on four seasonally based occasions. The morphology of AM root colonization had a similar pattern in the plants studied. Maximum arbuscular colonization occurred at the beginning of the growing season in late winter and was minimal in late summer, but maximal vesicular colonization occurred in summer and was minimal in winter, suggesting a preferential production of these morphological phases by the fungus with respect to season. The greatest arbuscular colonization was associated with the highest N and P concentrations in plant tissue, suggesting a correspondence with increases in the rate of nutrient transfer between the symbiotic partners. Water content, salinity and sodicity in soil were positively associated with AM root colonization and arbuscule colonization in L. tenuis , but negatively so in the grasses. There were distinct seasonally related effects with respect to both spore density and AM colonization, which were independent of particular combinations of plant species and soil sites.     

Cellophane surface-induced gene,VdCSIN1, regulates hyphopodium formation and pathogenesis via cAMP-mediated signalling in Verticillium dahliae

The soil-borne vascular pathogen Verticillium dahliae infects many dicotyledonous plants to cause devastating wilt diseases. During colonization, V. dahliae spores develop hyphae surrounding the roots. Only a few hyphae that adhere tightly to the root surface form hyphopodia at the infection site, which further differentiate into penetration pegs to facilitate infection. The molecular mechanisms controlling hyphopodium formation in V. dahliae remain unclear. Here, we uncovered a cellophane surface-induced gene (VdCSIN1) as a regulator of V. dahliae hyphopodium formation and pathogenesis. Deletion of VdCSIN1 compromises hyphopodium formation, hyphal development and pathogenesis. Exogenous application of cyclic adenosine monophosphate (cAMP) degradation inhibitor or disruption of the cAMP phosphodiesterase gene (VdPDEH) partially restores hyphopodium formation in the VdΔcsin1 mutant. Moreover, deletion of VdPDEH partially restores the pathogenesis of the VdΔcsin1 mutant. These findings indicate that VdCSIN1 regulates hyphopodium formation via cAMP-mediated signalling to promote host colonization by V. dahliae.