Trichoderma spp. as elicitors of wheat plant defense responses against Septoria tritici

Leaf blotch of wheat is a widespread and highly active disease that affects wheat production. In addition to the use of chemicals and proper cultivation methods, microbial antagonists are used to control plant pathogens. Trichoderma spp. stimulate a systemic induced response in plants. Therefore, the efficacy of Trichoderma spp. against wheat leaf blotch was evaluated under greenhouse conditions. The susceptible plants were sprayed with Septoria tritici conidiospores. In order to select an efficient method of pretreatment with Trichoderma spp., leaf spraying and seed coating with 14 isolates were tested in 2003 and 2004. The extent of leaf necrosis area and pycnidial coverage was estimated. Antagonism was assessed by the capacity of each Trichoderma spp. isolate to restrict the progress of leaf blotch, 21 days after inoculation. Of the two methods, seed coating was more efficacious against leaf blotch than leaf spraying. Amongst the 14 isolates tested, the isolate prepared from T. harzianum (Th5) produced the highest level of protection. None of the treatments caused changes in plant stem diameter or dry weight. Trichoderma spp. did not get into leaves while S. tritici was present, even in asymptomatic leaf extracts. In addition, the leaf apoplast antifungal proteolytic activity was measured in plants 7, 15, and 22 days after sowing. This antifungal action decreased in plants only inoculated with S. tritici, but increased in those grown from seeds coated with the T. harzianum (Th5) isolate. This increase conferred resistance to the susceptible wheat cultivar. The endogenous germin-like protease inhibitor coordinated the proteolytic action. These results suggest that T. harzianum stimulates a biochemical systemic induced response against leaf blotch.     

Synergistic effects of antagonistic fungi and a plant growth promoting rhizobacterium, an arbuscular mycorrhizal fungus, or composted cow manure on populations of Meloidogyne incognita and growth of tomato

Effects of four antagonistic fungi (Paecilomyces lilacinus, Pochonia chlamydosporia, Trichoderma harzianum and Gliocladium virens) alone and together with a plant growth promoting rhizobacterium Pseudomonas putida, an arbuscular mycorrhizal fungus Glomus intraradices or with composted cow manure (CCM) were assessed on the growth of tomato and on the reproduction of Meloidogyne incognita in glasshouse experiments. Application of all antagonistic fungi (except G. virens), P. putida, G. intraradices or CCM caused a significant increase in the growth of plants without nematodes. However, use of either of these fungi, P. putida, G. intraradices and CCM against plants with nematodes caused a significant increase in tomato growth. Paecilomyces lilacinus caused a 42% increase in the growth of nematode-inoculated plants followed by P. chlamydosporia (36%), T. harzianum (18%) and G. virens (15%). CCM caused about 57% increase in the growth of nematode-inoculated plants followed by P. putida (37%) and G. intraradices (31%). Maximum increase (71%) in the growth of nematode-inoculated plants was observed when CCM was used with P. lilacinus. Moreover, P. lilacinus caused a high reduction (55%) in galling and nematode multiplication, while G. virens the least (25%). Use of P. putida also caused a 39% reduction in galling and nematode multiplication followed by CCM (34%) and G. intraradices (32%). Combined use of CCM with P. lilacinus caused maximum reduction (79%) in galling and nematode multiplication. Re-isolation of antagonistic fungi from nematodes revealed that P. lilacinus parasitised more females and eggs than other antagonistic fungi. Root colonisation by P. putida was increased with P. lilacinus, while colonisation by G. intraradices was reduced in the presence of antagonistic fungi.     

Biocontrol of Pythium tracheiphilum in Chinese Cabbage by Clonostachys rosea under Field Conditions

Control of leaf and head rot of Chinese cabbage (Brassica campestris L. ssp. pekinensis), caused by Pythium tracheiphilum, was obtained by Clonostachys rosea (isolate IK726) in field trials conducted in 1995 and 1999 on naturally infested land in a commercial crop in Denmark. A significant 2-3-fold disease reduction was obtained at an application rate of 10⁸-10⁹ conidia m^-2 (high application rate) in both years, but not at a 10-fold reduced rate in 1999. Disease reduction by Trichoderma harzianum (Supresivit) was almost significant at the high application rate (1 g product m^-2 corresponding to 7×10⁹ colony forming units (CFUs) m^-2) in both years, but not at a 10-fold reduced rate applied in 1999. In both 1995 and 1999 trials, the percentage of marketable heads increased significantly by 10% following a full application rate of C. rosea. Supresivit applied at the full application rate gave a significant 13% yield improvement in 1995 but not in 1999. No yield improvement was found when the two agents were applied at 10-fold reduced rates. A Danish T. harzianum isolate significantly increased yield by 13% in 1995, but gave no disease control. Plant growth promotion may have been responsible for yield improvements obtained by Supresivit and the Danish isolate of T. harzianum. The 1995 trial also evaluated the products Binab T (T. harzianum+T. polysporum), Mycostop (Streptomyces griseoviridis), Polyversum (P. oligandrum) and Aliette (fosetyl-Al) and Danish isolates of P. oligandrum (2) and T. virens (1), none of which gave disease control or yield effects.     

The effect of Trichoderma harzianum and T. koningii on the control of tan spot (Pyrenophora tritici-repentis) and leaf blotch (Mycosphaerella graminicola) of wheat under field conditions in Argentina

The effect of six isolates of Trichoderma harzianum and one isolate of T. koningii on the incidence and severity of tan spot (Pyrenophora tritici-repentis) and leaf blotch of wheat (Mycosphaerella graminicola) was evaluated under field conditions. Significant differences between wheat cultivars, inoculum types and growth stages were found. Three of the isolates tested (T2 for M. graminicola, T7 for P. tritici-repentis and T5 for both of them) showed the best performance in controlling leaf blotch and tan spot when coated onto seed or sprayed onto wheat leaves at different growth stages, with significant severity reduction up to 56%. At tillering, six of the isolates reduced the severity of P. tritici-repentis and M. graminicola compared to the control by up to 39% and 12-53%, respectively. In some experiments, the biocontrol preparation (T2 and T5) gave a level of disease control similar to that obtained with Tebuconazole (70 and 48%, respectively). The effect of Trichoderma against P. tritici-repentis was also observed at the heading stage, when six of the treatments reduced disease severity by 16-35%. This is the first report on the efficacy of Trichoderma spp. against wheat necrotrophic pathogens under field conditions in Argentina.     

Phenotypic Diversity among Alleles at the PER-1 Locus of NEUROSPORA CRASSA

Comparison of 11 perithecial color mutants suggested that all were alleles at the per-1 locus but nonetheless separable into two groups because of phenotypic differences. Three of the mutant strains produced orange perithecia and black ascospores, and eight produced paler, yellow perithecia and white ascospores. Perithecial phenotype was dependent upon the genotype of the protoperithecial parent; ascospore phenotype, upon the genotype of the individual ascospore. No evidence was found that the white ascospores were due to chromosomal rearrangements. No separation of the perithecial and ascospore phenotypes by recombination was observed in a cross between one of the mutants and a per-1+ strain. However, apparent low levels of recombination in crosses between some of the mutants indicated possible genetic complexity at the per-1 locus. The phase specificity of the per-1 mutations and the possible nature and mode of expression of the orange and yellow perithecial pigments are discussed.     

Physiological and environmental aspects of ascospore discharge in Gibberella zeae (anamorph Fusarium graminearum)

We investigated ascospore discharge in the perithecial fungus, Gibberella zeae. In a wind tunnel study that simulated constant rain and varying day and night lengths, the rate of ascospore release was approximately 8-30% greater under light than in complete darkness. Under constant light, ascospore discharge occurred at maximal rates at relative humidity levels greater than 92%. When perithecia were placed under conditions of high external osmolarity, ascospore discharge was significantly reduced. Ascospores were discharged from asci along with droplets of fluid, the epiplasm, from within the ascus. Analysis of discharged epiplasmic fluid by GC-MASS Spectrometry revealed that mannitol was the major simple sugar component of the fluid. Activity of mannitol dehydrogenase, which catalyzes the conversion of fructose to mannitol, was higher in protein extracts from mature perithecia than in extracts from vegetative tissue. Several inhibitors of K(+) and Ca(++) ion channels inhibited ascospore discharge, which suggested that ascospore discharge resulted from the buildup of turgor pressure generated by ion fluxes and mannitol accumulation.     

板栗褐缘叶枯病协同致病菌Ophiognomonia castaneae的生活史

采用室外定点观察,子实体诱导及rDNA ITS、MS204、tef1-α 3种分子标记进行系统发育分析等方法,对板栗褐缘叶枯病Phomopsis castaneae-mollissimae的协同致病菌板栗蛇孢日规壳Ophiognomonia castaneae的生活史进行了研究。结果表明,每年7月下旬至8月初叶片发病初期很少分离到O. castaneae,随着病斑扩大该菌的分离频率逐渐增大,至发病后期其分离频率可高达78.5%,甚至可超过致病菌P. castaneae-mollissimae,10月下旬板栗落叶背面的病斑上开始形成O. castaneae的分生孢子盘,11月下旬开始形成O. castaneae的子囊壳原基,次年5、6月越冬落叶背面的病斑上长出子囊壳;带病斑的叶片经室内外诱导,0-25℃范围均可产生成熟子囊壳;湿度是决定子囊壳能否形成的关键因素,强光照不利于子囊壳的产生;分离物的菌丝体在PDA培养基上培养,易产生分生孢子;将分离物分为两种交配型,相互交配后6个月所有处理均未长出该菌的有性型子实体。室外定点观察及rDNA ITS、MS204、tef1-α 3种分子标记表明分离物和病斑上的子囊孢子及其萌发菌丝为O. castaneae的不同生长发育阶段。     

Application of response surface methodology to evaluate the influence of temperature and initial pH on the production of β-1,3-glucanase and carboxymethylcellulase from Trichoderma harzianum

Response surface methodology was employed to study the effect of temperature and initial pH on the production of β-1,3-glucanase and carboxymethylcellulase from Trichoderma harzianum in both surface culture and submerged culture experiments. The efficiency of the enzymes in generating protoplasts from Trichoderma reesei mycelium was also studied. Regression analysis was performed on the data obtained. A temperature of 30°C and an initial pH of 4.7 were found to be optimal for β-1,3-glucanase production from T. harzianum in both surface culture and submerged culture processes.     

Scanning electron microscopy observations of the interaction between Trichoderma harzianum and perithecia of Gibberella zeae

Chronological events associated with the interaction between a strain of Trichoderma harzianum, T472, with known biological control activity against perithecial production of G. zeae, were studied with scanning electron microscopy to investigate the mechanisms of control. Large clusters of perithecia consisting of 5-15 perithecia formed on the autoclaved, mulched wheat straw inoculated with G. zeae alone (control) with an average of 157 perithecia per plate. Small clusters consisting of 3-6 and an average of 15 perithecia per plate perithecia formed on straw that was treated with T. harzianum. The mature perithecia from straw treated with T. harzianum produced less pigment and were lighter in color than those from the control plates. Furthermore the cells of the outer wall of these perithecia were abnormal in appearance and unevenly distributed across the surface. Immature perithecia were colonized by T. harzianum approximately 15 d after inoculation (dai) with the biocontrol agent and pathogen. Few perithecia were colonized at later stages. The affected perithecia collapsed 21 dai, compared to the perithecia in the control samples that began to collapse 28 dai. Abundant mycelium of T. harzianum was seen on the perithecia of treated samples. Perithecial structures may be resistant to penetration by the mycelium because direct penetration was not observed. Trichoderma harzianum colonized the substrate quickly and out-competed the pathogen, G. zeae.     

Laboratory and glasshouse evaluation of entomopathogenic fungi against the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae), on tomato, Lycopersicon esculentum

Forty isolates of entomopathogenic fungi from six genera were assessed against the two-spotted spider mite, Tetranychus urticae, in a single dose, direct application laboratory bioassay on tomato leaflets. Only three isolates caused greater mortality than the control: these were Metarhizium anisopliae 442.99, Hirsutella spp. 457.99, and Verticillium lecanii 450.99. These isolates were assessed in a multiple dose bioassay, together with three isolates cultured from commercial biopesticides as follows: Beauveria bassiana 432.99 (cultured from 'Naturalis-L', Troy Biosciences, Phoenix, TX, USA); Hirsutella thompsonii 463.99 (cultured from 'Mycar', Abbott Laboratories USA); and V. lecanii 19.79 (used in 'Mycotal' Koppert BV, The Netherlands). Beauveria bassiana 432.99, H. thompsonii 463.99, M. anisopliae 442.99, and V. lecanii 450.99 were all pathogenic to T. urticae in this bioassay. In addition, it was found that the mortality caused by B. bassiana 432.99 and Naturalis-L was increased when the mites were exposed to tomato leaflets sprayed previously with conidia suspensions, compared to spraying the mites directly. In a glasshouse experiment, sprays of B. bassiana 432.99, H. thompsonii 463.99, M. anisopliae 442.99, V. lecanii 450.99 and Naturalis-L reduced T. urticae populations in a tomato crop grown according to commercial practice. Naturalis-L reduced T. urticae numbers by up to 97%. In a second glasshouse experiment, single sprays of Naturalis-L and the acaricide fenbutatin oxide (Torq) were compared as supplementary treatments to release of the predatory mite, Phytoseiulus persimilis. Supplementary sprays of fenbutatin oxide reduced the numbers of T. urticae nymphs (80% reduction), while Naturalis-L reduced numbers of T. urticae adults, nymphs and eggs (98% reduction in all three cases). It is concluded that Naturalis-L has the potential to be used against T. urticae on glasshouse tomato crops.     

木霉对月季幼苗生长的影响

为了研制促进月季生长的木霉生防菌肥，采用本实验室分离鉴定的哈茨木霉T6施于月季盆栽土壤中，分析木霉菌对"十姐妹"月季硬枝扦插发芽、幼苗生长、防御酶活性和内源激素水平的影响。结果表明：施用哈茨木霉T6的月季硬枝出芽率和幼苗枝条长度分别是对照的1.4和1.82倍；并且枝条和叶的干重、枝条和叶的含水率均显著增加。此外显著提高了叶中3种防御酶SOD、POD、CAT的活性及6种内源激素IAA、GA₃、ZT、ABA、SA和JA的水平。说明木霉T6具有促进月季生长和提高其抗性的作用，研究结果为研制促进月季生长的生物菌肥提供理论依据。     

Field and laboratory studies of the effect of urea on ascospore production of Venturia inaequalis (Cke.) Wint

Applications of urea after harvest but before leaf-fall restricted perithecial production by Venturia inaequalis. Immersion of detached leaves in urea appeared to be the most effective method of preventing perithecial formation, although spraying attached leaves was equally effective when leaf abscission occurred within a week of treatment. A high nitrogen content within the leaf was one of the major factors contributing to suppression. Urea-treated leaves decomposed rapidly, thus destroying the overwintering substrate for the fungus. When apple plants (clone M. 111) were sprayed in autumn with 5 % urea, followed by a second (pre-bud-burst) application at 2 %, ascospore production in the spring was suppressed. The second treatment appeared to prevent the release of ascospores from mature perithecia.     

产木聚糖酶厌氧真菌菌株筛选及产酶培养条件研究*

从12株分离自反刍动物瘤胃及粪样的厌氧真菌中筛选到一株木聚糖酶高产菌,编号为A4,初步鉴定为Neocallimastix属菌。以稻草秸、玉米秸、花生秸、滤纸片段为发酵底物,经39℃厌氧培养,A4菌产生的木聚糖酶活分别为14.31U/mL、11.39U/mL、6.99U/mL和13.38U/mL。对A4菌产生木聚糖酶的条件进行优化,结果发现,培养基中无细胞瘤胃液浓度对A4菌产生的木聚糖酶活无显著影响;但酵母膏浓度从1.0g/L降至0.5g/L后,A4菌产生的木聚糖酶活显著下降(P<0.05)。     

Nutrient-dependent inhibition of perithecial development due to sequential crosses on the same mycelium of Neurospora tetrasperma

Summary An explanation of perithecial inhibition in the second of two sequential crosses at different locations on the same mycelium of Neurospora tetrasperma was sought by (1) assaying media that had supported inhibited and uninhibited portions of the mycelium which contained no developing perithecia, (2) determining the effect of these media on perithecial development, (3) adding nutrients to inhibited portions of the mycelium, and (4) assaying carbon sources in media that had supported portions of the mycelium which contained developing perithecia, and portions, both inhibited and uninhibited, which contained no developing perithecia. Different kinds and volumes of media and various intervals of time between sequential crosses were used to aid in determining limits of perithecial inhibition. Perithecial inhibition was observed to be independent of volatile metabolites and pH, independent of non-volatile metabolites, reversible by addition of nutrients, dependent upon nutrient volume, and correlated with the concentration of the carbon source in the medium. It is proposed that second crosses are inhibited because of a previous lowering of the concentration of nutrients in the medium in second-cross locations, owing to prior demand upon those nutrients by the developing perithecia in first-cross locations. The possibility of an activation signal between first- and second-cross locations is discussed. No inhibitory substance in inhibited locations was detected.Supported in part by a National Science Foundation Traineeship.     

Effects of Temperature and Moisture on Development of Fusarium graminearum Perithecia in Maize Stalk Residues

Fusarium graminearum is the predominant component of the Fusarium head blight complex of wheat. F. graminearum ascospores, which initiate head infection, mature in perithecia on crop residues and become airborne. The effects of temperature (T) and moisture on perithecium production and maturation and on ascospore production on maize stalk residues were determined. In the laboratory, perithecia were produced at temperatures between 5 and 30°C (the optimum was 21.7°C) but matured only at 20 and 25°C. Perithecia were produced when relative humidity (RH) was ≥75% but matured only when RH was ≥85%; perithecium production and maturation increased with RH. Equations describing perithecium production and maturation over time as a function of T and RH (R² > 0.96) were developed. Maize stalks were also placed outdoors on three substrates: a grass lawn exposed to rain; a constantly wet, spongelike foam exposed to rain; and a grass lawn protected from rain. No perithecia were produced on stalks protected from rain. Perithecium production and maturation were significantly higher on the constantly wet foam than on the intermittently wet lawn (both exposed to rain). Ascospore numbers but not their dispersal patterns were also affected by the substrate.     

Genotypic variation among lineages of Trypanosoma cruzi and its geographic aspects

Isozyme analysis with 18 enzyme loci was conducted on 146 isolates of Trypanosoma cruzi from Mexico, Guatemala, Colombia, Ecuador, Peru, Brazil, Bolivia, Paraguay and Chile. Forty-four different MLGs (groups of isolates with identical multilocus genotypes) were identified and a phylogeny was constructed. The phylogenetic tree consisted of two main groups (T. cruzi I, T. cruzi II), and the latter was further divided into two subgroups (T. cruzi IIa, T. cruzi IIb–e). Evidence of hybridization between different MLGs of T. cruzi II was found, which means that genetic exchanges seem to have occurred in South American T. cruzi. On the other hand, the persistence of characteristic T. cruzi I and T. cruzi II isozyme patterns in single small villages in Bolivia and Guatemala suggested that genetic exchange is very rare between major lineages. A significant difference in genetic diversity was shown between T. cruzi I and T. cruzi II from several indices of population genetics. Two possibilities could explain this genetic variation in the population: differences in evolutionary history and/or different tendencies to exchange genetic material. Broad-scale geographic distributions of T. cruzi I and T. cruzi IIb–e were different; T. cruzi I occurred in Central America and south to Bolivia and Brazil, while T. cruzi IIb–e occurred in the central and southern areas of South America, overlapping with T. cruzi I in Brazil and Bolivia.     

木霉菌对芸芥生长相关生理指标的影响

芸芥(Eruca sativa)是当今我国蔬菜市场中一种颇具开发价值的新特芳香蔬菜。为了研制促进芸芥生长和提高品质的生防木霉菌剂,本研究以芸芥为试材,在大田条件下,采用本实验室分离鉴定的哈茨木霉T8进行浸种和浇根处理,分析木霉菌对一个生长季内连续栽培三茬30 d龄芸芥的生长相关生理指标的影响。结果表明,施用木霉菌能显著提高芸芥的生物量,改善其光合特性,增强其防御酶活性和脯氨酸含量,提高其产量和营养价值。     

Effects of Weather Variables on Ascospore Discharge from Fusarium graminearum Perithecia

Fusarium graminearum is a predominant component of the Fusarium head blight (FHB) complex of small grain cereals. Ascosporic infection plays a relevant role in the spread of the disease. A 3-year study was conducted on ascospore discharge. To separate the effect of weather on discharge from the effect of weather on the production and maturation of ascospores in perithecia, discharge was quantified with a volumetric spore sampler placed near maize stalk residues bearing perithecia with mature ascospores; the residues therefore served as a continuous source of ascospores. Ascospores were discharged from perithecia on 70% of 154 days. Rain (R) and vapor pressure deficit (VPD) were the variables that most affected ascospore discharge, with 84% of total discharges occurring on days with R≥0.2 mm or VPD≤11 hPa, and with 70% of total ascospore discharge peaks (≥ 30 ascospores/m³ air per day) occurring on days with R≥0.2 mm and VPD≤6.35 hPa. An ROC analysis using these criteria for R and VPD provided True Positive Proportion (TPP) = 0.84 and True Negative Proportion (TNP) = 0.63 for occurrence of ascospore discharge, and TPP = 0.70 and TNP = 0.89 for occurrence of peaks. Globally, 68 ascospores (2.5% of the total ascospores sampled) were trapped on the 17 days when no ascospores were erroneously predicted. When a discharge occurred, the numbers of F. graminearum ascospores sampled were predicted by a multiple regression model with R² = 0.68. This model, which includes average and maximum temperature and VPD as predicting variables, slightly underestimated the real data and especially ascospore peaks. Numbers of ascospores in peaks were best predicted by wetness duration of the previous day, minimum temperature, and VPD, with R² = 0.71. These results will help refine the epidemiological models used as decision aids in FHB management programs.     

New species from the Fusarium solani species complex derived from perithecia and soil in the old World tropics

A large collection of strains belonging to the Fusarium solani species complex (FSSC) was isolated from soil and perithecia in primary forests in Sri Lanka (from fallen tree bark) and tropical Australia (Queensland, from fallen tree fruits and nuts). Portions of the translation elongation factor 1-alpha (tef1) gene, the nuclear large subunit (NLSU) and internal transcribed spacer regions (ITS) of the nuclear ribosomal RNA genes were sequenced in 52 isolates from soil and perithecia. The FSSC was divided previously into three clades with some biogeographic structure, termed Clades 1, 2 and 3. All Sri Lankan and Australian soil isolates were found to be members of Clade 3, most grouping with the cosmopolitan soil-associated species F. falciforme. All but two Sri Lankan perithecial isolates were associated with a set of five divergent phylogenetic lineages that were associated with Clade 2. Australian perithecial isolates resided in a subclade of Clade 3 where most of the previously defined mating populations of the FSSC reside. Isolates from perithecia and those cultured from soil were always members of different species lineages, even when derived from proximal locations. The previous biogeographic assignment of Clade 2 to South America is now expanded to the worldwide tropics. Sri Lanka appears to be an important center of diversity for the FSSC. Nectria haematococca is epitypified with a collection from the type locality in Sri Lanka; its anamorph is described as a new species, Fusarium haematococcum. Neocosmospora E.F. Smith is adopted as the correct genus for Nectria haematococca. These new species are described: F. kurunegalense/Neo. kurunegalensis, F. rectiphorus/Neo. rectiphora/, F. mahasenii/Neo. mahasenii/, F. kelerajum/Neo. keleraja.     

Botryosphaeran, a new substrate for the production of β-1,3-glucanases by Botryosphaeria rhodina and Trichoderma harzianum Rifai

Botryosphaeria rhodina and Trichoderma harzianum Rifai were grown on botryosphaeran (an exopolysaccharide (EPS) of the β-1,3;1,6-d-glucan type produced by B. rhodina) as sole carbon source with the objective of producing β-glucanases of the 1,3-type. Conditions for β-1,3-glucanase production by T. harzianum were examined by a statistical response surface method, and showed maximal enzyme production at 5 days growth in media containing 1.5 g/l of EPS. Good agreement was obtained between the experimental values of β-1,3-glucanase activity and the corresponding values predicted by the mathematical model. The crude β-1,3-glucanase preparations were active towards a number of different β-1,3-glucans and β-glucosides. The mycelium of B. rhodina also proved to be a good substrate for β-1,3-glucanase production by both fungal species.