Investigation on the Fusarium induced stress protein (FISP) in wheat: Immunolocalisation in root cells

Abstract

Fusarium induced-stress-protein (FISP) of ～51 kDa molecular mass was detected in seven day old germinated wheat (Triticum aestivum var Sonalika) seedlings infected with F. oxysporum for a period of seven days. This particular stress protein (FISP) of ～51 kDa was over-expressed in the case of Fusarium infected seedlings compared to the untreated seedlings where the presence of this protein was insignificant. Localisation of this ～51 kDa protein in root tissue by anti-CSAP (Cadmium Stress Associated Protein) antiserum showed a significantly higher number of gold particles in the case of Fusarium infected root tissue compared to the untreated control. A unique type of organised localisation of FISP around the plasma membrane and outer vacuolar membrane suggests its defensive role against Fusarium infection that might be a general stress protein against biotic and abiotic stresses.     

Biocontrol of onion white rot by application of Trichoderma species formulated on wheat bran powder

In vitro, Trichoderma album, Trichoderma harzianum, Trichoderma koningii, Trichoderma viride and Trichoderma virens showed antagonistic effect against the most pathogenic isolate (Sc2) of Sclerotium cepivorum, the cause of onion white rot disease. Five Trichoderma preparations of each Trichoderma sp. were prepared on wheat bran powder to be used for controlling white rot disease of onion. Greenhouse and field experiments followed the same trend where T. harzianum and T. koningii were the most effective in reducing the incidence and severity of white rot disease compared with the control. Trichoderma species preparations caused promotion to vegetative parameters of onion plants in pots and increase bulb productivity in filed. In this regard, T. harzianum and T. koningii were the most effective. A positive correlation was found between the biocontrol activity of Trichoderma species preparations and enhancement of peroxidase, polyphenoloxidase and chitinase enzymes in onion plants to resist infection with S. cepivorum.     

Antagonistic activity and hyphal interactions of Trichoderma spp. against Fusarium proliferatum and F. oxysporum in vitro

Trichoderma is a well-known antagonist against soilborne plant pathogens. However, the species and even various isolates have different biocontrol potential. To evaluate the antagonistic activities of Trichoderma harzianum, T. harzianum strain T100 (T100), T. viride and T. haematum against Fusarium oxysporum and F. proliferatum, we used dual culture and productions of volatile and non-volatile metabolites in three different phases in vitro. An analysis of the data in dual culture tests represented T. viride, T. haematum and T100 as effective antagonists of Fusarium while T100 was the only fungus being able to lyse the confronting mycelia. Similar results were obtained in the volatile metabolites tests also. In contrast with the two previous tests, the non-volatile metabolites produced by T. harzianum inhibited Fusarium mycelial growth the most, and T100 acted moderately. It was also clearly showed that the antagonistic effect of Trichoderma spp. was more on F. proliferatum than on F. oxysporum. Finally, because Trichoderma spp. was most effective in the second phase, we recommend to use T100 against F. proliferatum at the initial stages of infection as its mycoparasitism on F. oxysporum was observed microscopically through forming apressoria structures without any coiling around the pathogen.     

Antagonistic assessment of Trichoderma spp. by producing volatile and non-volatile compounds against different fungal pathogens

Trichoderma spp. are well-known biological agents that have significant antagonistic activity against several plant pathogenic fungi. In the present study, Trichoderma spp. were tested in vitro for their antagonistic activity against different spp. of Fusarium and Alternaria viz. Alternaria alternata, A. brassicae, A. solani, Fusarium oxysporum and F. solani using dual plate assay and by the production of volatile and non-volatile compounds. The results obtained revealed that Trichoderma harzianum and T. viride effectively inhibited the growth and spore production of different spp. of Fusarium and Alternaria. The highest growth inhibition was found in A. alternata 62.50% and 60.00% by non-volatile compounds of T. harzianum and T. viride, respectively. Similarly, the volatile compounds inhibit the maximum growth of A. alternata 40.00% and 35.00% by T. harzianum and T. viride, respectively. Volatile and non-volatile compounds of Trichoderma spp. were analysed by GC-MS technique and the properties of distinguished compounds showed antifungal, antimicrobial and antibiotic activities. Volatile compounds of T. harzianum and T. viride showed highest percent abundance for glacial acetic acid (45.32%) and propyl-benzene (41.75%), respectively. In case of non-volatile compounds, T. harzianum and T. viride showed D-Glucose, 6-O-α-D-galactopyranosyl- (38.45%) and 17-Octadecynoic acid (36.23%), respectively. The results of present study confirmed that T. harzianum can be used as a promising biological control agent against Alternaria and Fusarium spp. that cause diseases in various vegetables and crops.     

Fusaric acid accelerates the senescence of leaf in banana when infected by Fusarium

Fusarium oxysporum f.sp. cubense (FOC) is a causal agent of vascular wilt and leaf chlorosis of banana plants. Chloroses resulting from FOC occur first in the lowest leaves of banana seedlings and gradually progress upward. To investigate the responses of different leaf positions to FOC infection, hydroponic experiments with FOC inoculation were conducted in a greenhouse. Fusarium-infected seedlings exhibited a decrease in net photosynthesis rate, stomatal conductance, and transpiration rate of all leaves. The wilting process in Fusarium-infected seedlings varied with leaf position. Measurements of the maximum photochemical efficiency of photosystem II (F _V/F _max) and visualization with transmission electron microscopy showed a positive correlation between chloroplast impairment and severity of disease symptoms. Furthermore, results of malondialdehyde content and relative membrane conductivity measurements demonstrated that the membrane system was damaged in infected leaves. Additionally, the activities of phenylalanine ammonia-lyase, peroxidase and polyphenol oxidase were increased and total soluble phenolic compounds were significantly accumulated in the leaves of infected plants. The structural and biochemical changes of infected plants was consistent with plant senescence. As the FOC was not detected in infected leaves, we proposed that the chloroplast and membrane could be damaged by fusaric acid produced by Fusarium. During the infection, fusaric acid was first accumulated in the lower leaves and water-soluble substances in the lower leaves could dramatically enhance fusaric acid production. Taken together, the senescence of infected banana plants was induced by Fusarium infection with fusaric acid production and the composition of different leaf positions largely contribute to the particular senescence process.     

Tissue‐specific and pathogen‐inducible expression of a fusion protein containing a Fusarium‐specific antibody and a fungal chitinase protects wheat against Fusarium pathogens and mycotoxins

Fusarium head blight (FHB) in wheat and other small grain cereals is a globally devastating disease caused by toxigenic Fusarium pathogens. Controlling FHB is a challenge because germplasm that is naturally resistant against these pathogens is inadequate. Current control measures rely on fungicides. Here, an antibody fusion comprised of the Fusarium spp.‐specific recombinant antibody gene CWP2 derived from chicken, and the endochitinase gene Ech42 from the biocontrol fungus Trichoderma atroviride was introduced into the elite wheat cultivar Zhengmai9023 by particle bombardment. Expression of this fusion gene was regulated by the lemma/palea‐specific promoter Lem2 derived from barley; its expression was confirmed as lemma/palea‐specific in transgenic wheat. Single‐floret inoculation of independent transgenic wheat lines of the T₃ to T₆ generations revealed significant resistance (type II) to fungal spreading, and natural infection assays in the field showed significant resistance (type I) to initial infection. Gas chromatography–mass spectrometry analysis revealed marked reduction of mycotoxins in the grains of the transgenic wheat lines. Progenies of crosses between the transgenic lines and the FHB‐susceptible cultivar Huamai13 also showed significantly enhanced FHB resistance. Quantitative real‐time PCR analysis revealed that the tissue‐specific expression of the antibody fusion was induced by salicylic acid drenching and induced to a greater extent by F. graminearum infection. Histochemical analysis showed substantial restriction of mycelial growth in the lemma tissues of the transgenic plants. Thus, the combined tissue‐specific and pathogen‐inducible expression of this Fusarium‐specific antibody fusion can effectively protect wheat against Fusarium pathogens and reduce mycotoxin content in grain.     

Natural Fusarium Grain Infection Level in Wheat, Barley and Oat after Early Application of Fungicides and Herbicides

In Norway, early application of fungicides against cereal leaf diseases (before Zadoks 60) is common practice amongst farmers. Whether this procedure has any effect on Fusarium infection of the mature grain has been little investigated. To evaluate effects on Fusarium grain infection, cereal grains were sampled during 1996, 1997 and 1998 from 12 field trials where early spraying against fungal diseases in spring wheat, spring barley and oats was carried out. Percentage infected grains and frequency of different Fusarium species was analysed in every grain sample. The effect of fungicides, glyphosate and postemergence herbicides on Fusarium grain infection was studied. Significant increase in Fusarium infection was detected in fungicide‐treated plots compared with untreated plots. Fusarium avenaceum and F. tricinctum were the most frequent species detected. The internal ranking of Fusarium species remained the same after spraying. No significant effects were found on the level of Fusarium infection after glyphosate treatment in autumn or herbicide treatment during the growing season.     

Extracellular Production of l-Lysine α-Oxidase in Wheat Bran Culture of a Strain of Trichoderma viride

A mold strain Y244-2 capable of producing l-lysine α-oxidase, a new enzyme catalyzing the α-oxidative deamination of l-lysine, was identified as Trichoderma viride. Among strains belonging to the genus Trichoderma tested, only Trichoderma viride Y244-2 produced the enzyme in wheat bran culture. The maximum enzyme production by the mold grown on wheat bran was observed after 10 and 14 days incubation with and without NaN0₃, respectively. Addition of NaN0₃, NH₄N0₃, adenine, purine nucleosides, l-histidine, glycine or l-glutamine to wheat bran stimulated the production of the enzyme. In the liquid culture, the enzyme was produced extracellulary under the aerobic conditions, although the production was much lower than that in the wheat bran culture.     

绿色木霉组蛋白去乙酰化酶编码基因TvRpd3在木霉生物防治作用中的功能分析

【目的】本文借助基因编辑技术在具有生物防治潜力的绿色木霉(Trichoderma viride)中敲除组蛋白去乙酰化酶编码基因TvRpd3,来研究TvRpd3基因及其编码蛋白在提高木霉病原菌拮抗能力中的作用。【方法】利用融合PCR和同源重组策略构建了TvRpd3基因缺失的突变菌株,通过对峙培养、表型观察、免疫组化检测、代谢组学分析等系统比较TvRpd3基因敲除前后菌株的组蛋白乙酰化修饰水平、次级代谢产物合成、病原菌拮抗能力以及田间防治效果等。【结果】与野生型菌株相比,缺失TvRpd3基因的木霉工程菌(?TvRpd3)对多种病原菌表现出了更强的对峙抑制效果,其所产的发酵液对小麦白粉病、烟草黑胫病和番茄枯萎病的防治效果分别提高了62.27%、57.45%和70.71%。同时,敲除TvRpd3基因也显著改变了木霉工程菌所产次级代谢产物的种类和产量,抗生性物质的产量大幅提高。【结论】绿色木霉TvRpd3基因及其介导的组蛋白乙酰化修饰在提高绿色木霉生物防治中起着重要作用。     

Effects of vanillin on the community structures and abundances of Fusarium and Trichoderma spp. in cucumber seedling rhizosphere

Soil microbial communities are crucial to the functioning of agricultural systems but little information is available on the effects of allelochemicals on soil microorganisms in vivo. Cucumber seedlings grown in soil were treated with different concentrations of vanillin (0.02–0.2?μmol/g soil), a phenolic compound with autotoxic activity. The community structures and abundances of Fusarium and Trichoderma spp. in cucumber rhizosphere were analyzed by polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis and quantitative PCR, respectively. Results showed that vanillin changed the community structures of Fusarium and Trichoderma spp. Vanillin decreased the number of bands of Fusarium spp., but increased the number of bands, Shannon–Wiener and evenness indices of Trichoderma spp. (p?Fusarium and Trichoderma spp. (p?Fusarium and Trichoderma spp., and that these two microbial groups showed different responses to vanillin.     

Fusant Trichoderma HF9 with enhanced extracellular chitinase and protein content

Strain improvement was carried out to obtain higher chitinase and protein by inter-specific protoplast fusion between Trichoderma harzianum and Trichoderma viride. Fusant HF9 and parental strains of Trichoderma were compared for chitinase and protein production. 1% of glucose, sucrose and fungal cell wall (Rhizoctonia solani), were used as carbon source for cultivation of Trichoderma and fungal cell wall was the best to induce chitinase and protein. Usage of 0.5% colloidal chitin for the fungal growth under aerated conditions at pH 6.5 and 28°C led to higher chitinase and protein production. In these conditions fusant Trichoderma HF9 in comparison with parent strains had 3-, 2.5- and 1.5-fold increase of total chitinase, specific chitinase and protein, respectively. SDS-PAGE analysis revealed that it had 9 major protein bands with up-regulation compared to parent strains. Amino acid analysis showed that protein of culture filtrate of T. harzianum, T. viride and fusant Trichoderma HF9 had 8, 6 and 10 amino acids, respectively. The results obtained suggested that fusant HF9 could be an integration of T. harzianum and T. viride through protoplast fusion.     

<i>Trichoderma</i>-Induced Improvement in Growth,Photosynthetic Pigments,Proline, and Glutathione Levels in <i>Cucurbita pepo</i> Seedlings under Salt Stress

Salt stress is one of the major abiotic stress in plants. However, traditional approaches are not always efficient in conferring salt tolerance. Experiments were conducted to understand the role of Trichoderma spp. (T. harzianum and T. viride) in growth, chlorophyll (Chl) synthesis, and proline accumulation of C. pepo exposed to salinity stress. There were three salt stress (50, 100, and 150 mM NaCl) lavels and three different Trichoderma inoculation viz. T. harzianum, T. viride, and T. harzianum + T. viride. Salt stress significantly declined the growth in terms of the shoot and root lengths; however, it was improved by the inoculation of Trichoderma spp. C. pepo inoculated with Trichoderma exhibited increased synthesis of pigments like chl a, chl b, carotenoids, and anthocyanins under normal conditions. It was interesting to observe that such positive effects were maintained under salt-stressed conditions, as reflected by the amelioration of the salinity-mediated decline in growth, physiology and antioxidant defense. The inoculation of Trichoderma spp. enhanced the synthesis of proline, glutathione, proteins and increased the relative water content. In addition, Trichoderma inoculation increased membrane stability and reduced the generation of hydrogen peroxide. Therefore, Trichoderma spp. can be exploited either individually or in combination to enhance the growth and physiology of C. pepo under saline conditions.     

Production of laccases by Pleurotus ostreatus in submerged fermentation in co‐culture with Trichoderma viride

Aims: To evaluate the production and stability of laccases by Pleurotus ostreatus in liquid co‐cultures with Trichoderma viride as a function of infection time and agitation rate. Methods and Results: Pleurotus ostreatus cultures were infected with T. viride spores at 30 and 48 h. Maximal laccase volumetric activity was seen after 48 h (control cultures) or 72 h (co‐cultures) of cultivation time. Only the cultures infected at 30 h showed an increased laccase volumetric activity compared to control cultures. After maximal laccase volumetric activity value was reached, a sharp decrease in it was observed in control cultures. Co‐cultures exhibited a comparatively lower loss of activity. The influence of P. ostreatus and/or T. viride on the stability of laccase volumetric activity and isoenzyme pattern was evaluated. Trichoderma viride induced changes in the laccase isoenzyme pattern. Agitated cultures increased biomass growth and specific productivity threefold and sevenfold, respectively, to the static cultures. Conclusions: The laccase volumetric activity is very likely the result of the balance between biosynthesis and degradation/biotransformation rates occurring during the cultures. The individual presence of P. ostreatus or T. viride in the culture negatively affected the volumetric laccase activity. Significance and Impact of the Study: The evaluation of culture parameters that could influence Trichoderma–basidomycetes interaction and laccase production during submerged fermentation has not been reported. This study showed how laccase production in co‐cultures of P. ostreatus and T. viride was influenced by the infection time and agitation/oxygenation conditions.     

Salicylic acid induces resistance to <Emphasis Type="Italic">Septoria nodorum</Emphasis> berk. in wheat

The effect of salicylic acid (SA) on oxalate oxidase and peroxidase activities and hydrogen peroxide (H₂O₂) production in leaf cells has been studied in wheat of the susceptible cultivar Zhnitsa infected by Septoria nodorum, a pathogen of wheat leaf blotch. The results show that fungal hyphae spread into interstices between mesophyll cells and that infected tissues contain H₂O₂. Treatment with SA results in enhanced H₂O₂ production in mesophyll cells, which is due to activation of oxalate oxidase and peroxidase in the cell wall. It is proposed that the modulating effect of SA on oxidoreductase activities is involved in the induction of protective response to fungal infection in wheat plants.     

Effect of Helminthosporic Root Rot on the Lipid Content in Wheat Seedlings

The concentration of dry matter and the content of esterified fatty acids in total lipids of roots and etiolated shoots of 3- to 10-day-old seedlings of wheat (Triticum aestivum L.) infected with Bipolaris sorokiniana (Sacc.) Schoemaker, the agent of helminthosporic root rot, were determined in the course of germination. At the onset of germination, fungal infection caused a considerable increase in the dry matter concentration in both roots and shoots due to the enhanced mobilization of seed reserves. However, after the 7th day of germination, dry matter concentration fell below the level of noninfected control seedlings as a result of infection. The content of total lipids rose immediately after infection and always exceeded the control index up to the end of germination, in spite of a continuous decrease in this index in both control and infected seedling. It is concluded that an increase in the content of cellular lipids is a characteristic response of both shoots and roots to the root rot infection of wheat seedlings.     

Arbuscular mycorrhizal fungi and Trichoderma viride mediated Fusarium wilt control in tomato

The biocontrol potential of two arbuscular mycorrhizal fungi (AMF) (Funneliformis mosseae and Acaulospora laevis) and Trichoderma viride was assessed against tomato wilt caused by Fusarium oxysporum Schlecht. f. sp. lycopersici under pot condition. All the bioagent showed appreciable results in increasing plant growth. Combined inoculation of F. mosseae, A. laevis and T. viride showed maximum increases in plant height, shoot fresh weight, root dry weight, number of leaves and number of branches per plant while dual inoculation of F. mosseae and T. viride increased rest of the growth parameters like shoot dry weight, root fresh weight, root length and leaf area. AM colonisation and spore number was found highest in single inoculation of AMF, which decreases with the addition of T. viride. But, this decrease has no effect on biocontrol efficiency of bioagents. Photosynthesis, chlorophyll content and nutrient content were markedly decreased by pathogen infection. Bioagent application overcomes this effect and a remarkable increase in the plant phosphorus and nitrogen content was recorded. Among both the AMF, F. mosseae proved to be more effective strain compared to A. laevis for tomato. Maximum reduction in disease incidence and severity was recorded in combined inoculation of F. mosseae, A. laevis and T. viride. Whereas control plants without any bioagent showed maximum occurrence of disease. The findings of this study concludes that soil inoculation with F. mosseae along with root inoculation with conidial suspension of T. viride before transplantation offered better survival and resistance to tomato seedlings against Fusarium wilt.     

Seed biopriming with drought tolerant isolates of Trichoderma harzianum promote growth and drought tolerance in Triticum aestivum

Green house study was aimed to investigate the effect of seed biopriming with drought tolerant isolates of Trichoderma harzianum, viz. Th 56, 69, 75, 82 and 89 on growth of wheat under drought stress and to explore the mechanism underlying plant water stress resilience in response to Trichoderma inoculation. Measurements of relative water content, osmotic potential, osmotic adjustment, leaf gas exchange, chlorophyll fluorescence and membrane stability index were performed. In addition, analysis of the phenolics, proline, lipid peroxidation and measurements of phenylalanine ammonia‐lyase activity were carried out. Seed biopriming enhanced drought tolerance of wheat as drought induced changes like stomatal conductance, net photosynthesis and chlorophyll fluorescence were delayed. Drought stress from 4 to 13 days of withholding water induced an increase in the concentration of stress induced metabolites in leaves, while Trichoderma colonisation caused decrease in proline, malondialdehyde (MDA) and hydrogen peroxide (H₂O₂), and an increase in total phenolics. A common factor that negatively affects plants under drought stress conditions is accumulation of toxic reactive oxygen species (ROS), and we tested the hypothesis that seed biopriming reduced damages resulting from accumulation of ROS in stressed plants. The enhanced redox state of colonised plants could be explained by higher l ‐phenylalanine ammonia‐lyase (PAL) activity in leaves after 13 days of drought stress in Trichoderma treated plants. Similar activity was induced in untreated plants in response to drought stress but to a lower extent in comparison to treated plants. Our results support the hypothesis that seed biopriming in wheat with drought tolerant T. harzianum strains increased root vigour besides performing the process of osmoregulation. It ameliorates drought stress by inducing physiological protection in plants against oxidative damage, due to enhanced capacity to scavenge ROS and increased level of PAL, a mechanism that is expected to augment tolerance to abiotic stresses.     

Induction of PR proteins and resistance by the biocontrol agent Clonostachys rosea in wheat plants infected with Fusarium culmorum

Clonostachys rosea (CR) is a common worldwide saprophyte with destructive effect against several plant pathogenic fungi showing antagonistic features against a wide variety of pathogens. We recently isolated a strain of C. rosea, named CR47, from wheat crown infected with Fusarium culmorum (FC); this strain proved to be effective against Fusarium seed borne diseases of cereals under field condition. In this paper the function of C. rosea applied as seed treatment on wheat seedling growth was investigated. In addition, we investigated the expression pattern of peroxidases and chitinases as well as PR4 proteins following both CR treatments of seeds and FC infection and also in the three-component system pathogen–antagonist–wheat. Several chitinase isoforms were induced by CR-treatment both in coleoptiles and roots, whereas some peroxidase isoforms were induced only in the presence of both antagonist and pathogen. In the latter case, it seems that CR-treatment by itself promotes plant growth and reduces the peroxidase expression, while enhances some chitinase isoforms probably involved in cell wall disruption. Moreover, both the antagonist and the pathogen studied induced PR4 protein expression, which probably exerts its role on the invading microorganisms by a translation-inhibitory process that could be ascribed to their ribonuclease activity.     

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.     

Efficacy ofTrichoderma chitinases againstRhizoctonia solani, the rice sheath blight pathogen

Thirty-five strains ofTrichoderma viride andT. harzianum were screened for their antagonistic ability against the rice sheath blight pathogen,Rhizoctonia solani. The strains that inhibited/overgrew the phytopathogenic fungus were considered effective. Light microscopic studies showed the antagonism of the hyphae of effectiveTrichoderma strains towards their host hyphae. Chitinase activity ofTrichoderma culture filtrates was enhanced, when colloidal chitin was used as the sole carbon source, instead of glucose. Chitinase pattern differed among the four select strains. The chitinase isoforms are induced differentially by carbon sources. The chitin affinity column fraction ofTrichoderma culture filtrate inhibited,in vitro, the growth ofR. solani.