Proteomic study of biocontrol mechanisms of Trichoderma harzianum ETS 323 in response to Rhizoctonia solani

To elucidate the entire range of proteins that are secreted by Trichoderma harzianum ETS 323 in its antagonism with Rhizoctonia solani, an in vivo interaction between them was mimicked and not only the secreted cell wall-degrading enzymes (CWDEs) but also all of the proteome were investigated. Seven CWDEs, chitinase, cellulase, xylanase, beta-1,3-glucanase, beta-1,6-glucanase, mannanase, and protease,were revealed by activity assay, in-gel activity stain, 2-DE, and LC-MS/MS analysis. Extracellular protein extracts from media that contained R. solani exhibited much higher CWDE activities than media that did not contain R. solani. Cellulase and mannanase activity, however, were insignificant. Activity stain also revealed that beta-1,3-glucanase, beta-1,6-glucanase, and xylanase activity occurred exclusively in media that contained R. solani. Furthermore, 35 of the 43 excised spots on the 2-DE gel were successfully analyzed by LC-MS/MS, and eight proteins were identified. They were two glycoside hydrolases, two proteases, two beta-glucosidases, one endochitinase and, interestingly, one amino acid oxidase. Additionally, a possible mechanism was proposed to elucidate how the cell walls of R. solani are systematically enveloped and disintegrated.     

Relationship between the biocontrol fungus Trichoderma harzianum and the phytopathogenic fungus Fusarium solani f.sp. pisi

Relationship between the biocontrol fungus Trichoderma harzianum ThzID1-M3 and the plant pathogen Fusarium solani f.sp. pisi (Fsp) was determined in this study. Dual culture assay indicated the competitive interaction between ThzID1-M3 and Fsp. Alginate pellets of ThzID1-M3 and Fsp conidia were added to non-sterile soil with pea seeds. The interaction between Trichoderma and Fsp adversely affected their establishment in soil. The addition of ThzID1-M3 significantly reduced the Fsp population, as well as the colonization of roots by Fsp. In contrast, the added Fsp significantly reduced the proliferation of ThzID1-M3 and Trichoderma spp. The results suggest that the competition may be the main mechanism of the antagonistic activity of Trichoderma against Fsp.     

Volatile compounds emitted by sclerotia of Sclerotinia minor,Sclerotinia sclerotiorum,and Sclerotium rolfsii

Volatile compounds emitted by sclerotia of Sclerotinia minor, Sclerotinia sclerotiorum, and Sclerotium rolfsii were identified by solid phase microextraction followed by gas chromatography and mass spectometry. Both S. minor and S. sclerotiorum emitted 2-methylenebornane and 2-methylisoborneol. In addition, S. minor emitted mesityl oxide, gamma-butyrolactone, cis- and trans-linalool oxide, linalool, and trans-nerolidol. S. sclerotiorum emitted 2-methyl-2-bornene, 1-methylcamphene, and a diterpene with a molecular weight of 272. Sclerotium rolfsii did not emit any of these compounds but did emit delta-cadinene and cis-calamenene. Chemicals emitted by S. minor and S. sclerotiorum were tested to determine if they could stimulate germination of conidia of Sporidesmium sclerotivorum, a mycoparasite on sclerotia of Sclerotinia spp. Chemicals were tested at 1 part per billion to 100 parts per million, both in direct contact with conidia and near, but not in, physical contact. None of the chemicals alone nor a combination of all chemicals induced germination of conidia of S. sclerotivorum.     

菊花菌核病病原菌鉴定、抗性筛选与防治

菌核病是菊花生产中的一种重要病害。为明确菊花菌核病病原菌的种类、品种抗性差异和有效的防治药剂,本研究采用组织分离法对病原菌进行分离培养,结合形态学特征、生物学特征和分子生物学研究进行鉴定,并筛选了33个切花菊品种以及7种杀菌剂。结果表明,从感病植株上分离得到的病原菌能够重新感染菊花,并且再次分离得到的菌株与原菌株相同。通过病原菌的形态、感染特征和分子序列分析,确定了该病原菌为核盘菌(Sclerotinia sclerotiorum)。病原菌在酵母浸出粉胨葡萄糖培养基(YEPD)上的生长速度最快,在20~30℃时生长最好,在pH值5~7时生长较快,对碳源的种类要求不严格,以蛋白胨为氮源时生长较好。共筛选了33份菊花材料,未发现免疫及抗病品种,有6份中抗品种,13份中感品种,14份感病品种,且离体与活体接种差异性不大。氟酰胺和肟菌酯对核盘菌具有显著抑制效果,半数效应浓度(EC;)分别为0.09 mg·L^-1和0.65 mg·L^-1;此外,通过接种后喷施杀菌剂发现,氟酰胺和肟菌酯的防治效果分别达到了98.26%和90.93%,显著优于其他杀菌剂。本研究建立了菊花菌核病的鉴定、筛选与防治的基本方法,为后续菊花抗性育种与品种改良奠定了基础。     

河套灌区向日葵菌核病发生程度预测预报

根据2000-2009年河套灌区向日葵菌核病中心病株出现期、始盛期和发生程度资料与同期气象资料进行相关性分析,研究其与气象条件的关系并建立相应的预测方程。结果表明,降水量是影响向日葵菌核病发生的关键因子,温度、湿度、蒸发量、大风日数和日照时数也对菌核病发生时间和发生程度有重要影响。通过逐步回归,建立了分别于11月和翌年5月进行预报的6个模型,均通过了0.05水平的显著性检验,对历史资料的拟合效果较好;通过2010、2011年试报,预报值与实际值相差较小,可投入实际运行。     

Biological control of the root-knot nematode Meloidogyne javanica by Trichoderma harzianum

The filamentous fungi Trichoderma spp. is currently developed as biocontrol agents against many plant pathogens. Recent studies have shown that these fungi are able to infect nematode eggs and juveniles. In this research, biological control of root-knot nematode (Meloidogyne javanica) by Trichoderma harzianum BI was investigated in greenhouse and laboratory experiments. Results showed that different concentrations (10²–10⁸ spores/ml) of T. harzianum BI decreased nematode infection and other parameters significantly, compared to control. T. harzianum BI was able to penetrate nematode egg mass matrix and significantly decreased nematode egg hatching level. Specific activities of resistance-related enzymes, namely peroxidase (POX), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) increased significantly in T. harzianum BI inoculated plants. Maximum activities of POX, PPO and PAL were observed at the 5, 5 and 6 days after inoculation, respectively. Chitinase activity was also increased in culture filtrates of T. harzianum BI grown on wheat bran moistened with salt solution supplemented with colloidal chitin or nematode eggs. Maximum activity of chitinase was recorded at the 4 days after inoculation, in media supplemented with colloidal chitin (1.15 U/min per ml) and nematode eggs (0.85 U/min per ml). Results suggested that direct parasitism of eggs through the increase in extracellular chitinase activity, which would be indicator of eggs infection capability, and inducing plant defense mechanisms leading to systemic resistance are two main suppression mechanisms used by T. harzianum BI against nematode.     

Involvement of pachybasin and emodin in self-regulation of Trichoderma harzianum mycoparasitic coiling

Our aim was to determine the effects of two secondary metabolites secreted by Trichoderma harzianum, pachybasin and emodin, on the mycoparasitic coiling behavior and cAMP content of T. harzianum. The number of T. harzianum coils around Nylon 66 fiber was increased in the presence of R. solani. The number of T. harzianum coils around R. solani hyphae and Nylon 66 fiber were significantly increased in the presence of pachybasin and emodin. The cAMP level in T. harzianum was significantly increased by close contact with R. solani and much higer cAMP level in the presence of exogenous pachybasin and emodin. A cAMP inhibitor diminished the effect of pachybasin and emodin on T. harzianum coiling around Nylon 66 fiber. The results suggest that pachybasin and emodin mediate the increase in the number of Trichoderma mycoparasitic coils via cAMP signaling. This is the first report to suggest that pachybasin and emodin play roles in the biocontrol mechanism of Trichoderma.     

Improvement of Sclerotinia sclerotiorum resistance in Brassica napus by using B. oleracea

Sclerotinia stem rot is one of the most serious diseases in rapeseed (Brassica napus) due to the lack of resistance sources. A high level of resistance was reported in Brassica oleracea cytodeme, one of parental species of rapeseed. In this study, a panel of 55 resynthesized lines of B. napus (RS lines) derived from seven wild and two cultivated types of B. oleracea was evaluated for Sclerotinia resistance over 2 years. Relative to ‘Zhongyou 821’, a cultivar of B. napus with partial resistance against S. sclerotiorum, RS lines exhibited stronger stem resistance. Although the resistant level of RS lines was lower than that of corresponding parental B. oleracea, a moderate correlation between resistance of RS line and corresponding parental B. oleracea type was found both for leaf (r = 0.74, P = 0.02) and stem (r = 0.69, P = 0.04). Our data suggests that the RS lines are important resources to improve Sclerotinia resistance of current rapeseed. A breeding strategy is discussed to enhance the Sclerotinia resistance of rapeseed by using B. oleracea.     

Calcium deficiency and gibberellic acid enhance susceptibility of pumpkin and sunflower seedlings to Sclerotinia sclerotiorum infection

Pumpkin (Cucurbita pepo L.) and sunflower (Helianthus annuus L.) seedlings were grown in culture deficient in calcium and inoculated with Sclerotinia sclerotiorum (Lib.) de Bary. Three days after inoculation the seedlings showed a susceptibility to the pathogen that was inversely proportional to the calcium content of the nutrient solution. Plants treated with gibberellic acid (GA₃) became more susceptible to infection by S. sclerotiorum. Increased addition of GA₃ resulted in increases in infection both in rate and severity. Sunflower was more susceptible to the pathogen than was pumpkin. GA₃ treatment consistently decreased the calcium content in both species. The effect of calcium deficiency on infection by S. sclerotiorum was greatly amplified by GA₃ addition.     

Proteome changes in leaves of Brassica napus L. as a result of Sclerotinia sclerotiorum challenge

Sclerotinia stem rot, caused by the necrotrophic fungal pathogen Sclerotinia sclerotiorum, is a serious disease of canola (Brassica napus L.). To increase the understanding of the B. napus- S. sclerotiorum interaction, proteins potentially involved in mediating this interaction were identified and characterized. Upon infection of canola leaves by S. sclerotiorum, necrosis of host leaves was observed by 12 h and rapidly progressed during the later time points. These morphological observations were supported by microscopic study performed at different time points after pathogen challenge. Leaf proteins were extracted and analyzed by 2-DE, which revealed the modulation of 32 proteins (12 down- and 20 up-regulated). The identities of these proteins were established by ESI-q-TOF MS/MS and included proteins involved in photosynthesis and metabolic pathways, protein folding and modifications, hormone signaling, and antioxidant defense. Gene expression analysis of selected genes was performed by qRT-PCR, whereas the elevated levels of the antioxidant enzymes peroxidase and superoxide dismutase were validated by enzyme assays. To the authors' best knowledge, this is the first proteomics-based investigation of B. napus-S. sclerotiorum interaction, and the roles of many of the proteins identified are discussed within the context of this pathosystem.     

Effects of the introduction of a biocontrol strain of Trichoderma atroviride on non target soil micro-organisms

The main objective of this study was to assess the impact of the application of an antagonistic strain of Trichoderma atroviride on the native microbial soil communities. The structures of the fungal and bacterial communities were assessed by T-RFLP (terminal restriction fragment length polymorphism) method, based on T-RFLP analysis of 18S and 16S rRNA genes, respectively. Results showed that the introduction of the strain I-1237 into two soils slightly modified the microbial diversity, only for a short period of time. Nine months post-inoculation resilience took place, resulting in similar structures of the fungal and bacterial communities in the inoculated and control soils.     

Soil and compost humic fractions regulate the response of Sclerotinia sclerotiorum to exogenously added allelochemical compounds

Allelochemical compounds released by plants to signal their presence and needs interact in soils with very important macromolecules, such as humic acids (HAs), which are able to modulate the ultimate effects on target organisms. Most of the available studies on plants and microorganisms report the effects of allelochemicals or those of humic fractions, separately. In this study, we investigated the combined activity of these two types of compounds on the soil-resident fungus Sclerotinia sclerotiorum. Thus, ferulic acid (FA), caffeic acid (CA), benzoic acid (BA), salicylic acid (SA), gallic acid (GA) and phthalic acid (PA), exogenously applied to the fungal growth medium, were tested both alone and in combination with a soil HA (SHA) and a compost HA (CHA). The two HAs were also tested alone on the fungus. When the allelochemicals were applied alone, only FA, BA and SA evidenced a significant inhibition of mycelial growth, whereas FA, BA and CA increased the number of sclerotia formed. The two HAs alone reduced the early growth of the fungus and markedly stimulated sclerotia formation. A significant attenuation or, in some cases, suppression of the allelochemical effect on mycelial growth was caused by the coexistence in the medium of the allelochemical and each HA, especially CHA. Moreover, in general, the combinations of HA-allelochemical significantly stimulated sclerotia formation, with respect to the sole allelochemical, but decreased it with respect to HA alone. Thus, investigations on the response of fungi to plant-released allelochemicals should not exclude interactive aspects of these compounds with ubiquitous coexisting humic macromolecules.     

Detection of Sclerotinia sclerotiorum using a monomeric and dimeric single-chain fragment variable (scFv) antibody

Sclerotinia sclerotiorum (Lib.) de Bary is a phytopathogenic fungus capable of causing significant yield losses in numerous crops, including canola, in which the fungus causes sclerotinia stem rot. Immunological detection methods to rapidly determine the presence of S. sclerotiorum on plants may provide growers with a viable diagnostic tool to aid with fungicide use decisions. This paper discusses the generation of a monomeric and dimeric single-chain, variable fragment (scFv) antibody with affinity for S. sclerotiorum using phage display technology. The bacterially expressed and purified scFv is shown to bind S. sclerotiorum with some cross-reactivity with the closely related phytopathogen Botrytis cinerea (Pers.:Fr.). The dimeric scFv displayed improved binding to the fungus as compared to the monomer and could detect the presence of mycelia in inoculated canola petals. To the authors' knowledge, this is the first report of a scFv dimer with affinity for S. sclerotiorum that has the potential for use in the development of a new diagnostic test.     

A novel L-amino acid oxidase from Trichoderma harzianum ETS 323 associated with antagonism of Rhizoctonia solani

Trichoderma spp. are used as biocontrol agents against phytopathogens such as Rhizoctonia solani, but their biocontrol mechanisms are poorly understood. A novel L-amino oxidase (Th-LAAO) was identified from the extracellular proteins of Trichoderma harzianum ETS 323. Here, we show a FAD-binding glycoprotein with the best substrate specificity constant for L-phenylalanine. Although the amino acid sequence of Th-LAAO revealed limited homology (16-24%) to other LAAO members, a highly conserved FAD-binding motif was identified in the N-terminus. Th-LAAO was shown to be a homodimeric protein, but the monomeric form was predominant when grown in the presence of deactivated Rhizoctonia solani. Furthermore, in vitro assays demonstrated that Th-LAAO had an antagonistic effect against Rhizoctonia solani and a stimulatory one on hyphal density and sporulation in T. harzianum ETS 323. These findings further our understanding of T. harzianum as a biocontrol agent and provide insight into the biological function of l-amino acid oxidase.     

钩状木霉和哈茨木霉菌对人参土壤理化性质及真菌群落结构的影响

为研究不同木霉菌对栽培人参土壤的改良作用,分析木霉菌对土壤养分和土壤真菌群落结构与功能特征的影响。采用人参盆栽试验,添加外源木霉菌（钩状木霉DH3、MM3、MM4、Q7 4种菌株和哈茨木霉MM6菌种）后,分析土壤理化性质以及土壤微生物群落多样性变化。添加不同外源木霉菌处理后人参土壤理化性质发生变化,土壤pH值无显著变化,土壤全氮、有机质、铵态氮、硝态氮、有效磷、速效钾含量显著增加（P<0.05）,而碳氮比（C/N）在添加木霉菌处理中降低,其中钩状木霉MM3和哈茨木霉MM6两株菌的理化性质表现最好。ITS测序分析获得703个OTUs,涵盖了8门、26纲、46目、86科、141属。木霉菌处理后土壤真菌微生物α多样性指数（ACE、Chao1）增加,尤其是钩状木霉MM3;钩状木霉MM3中的主要特征种群是肉座菌科（Hypocreaceae）和木霉菌属（Trichoderma）,哈茨木霉MM6的主要特征种群是毛壳菌科（Chaetomiaceae）。冗余分析（RDA）发现,土壤C/N和硝态氮是影响土壤环境中微生物多样性的主要因素;相关分析表明,刺球菌属（Chaetosphaeria）与土壤全氮...     

Interactive effects of Nemarioc-AL phytonematicide,Steinernema feltiae and Trichoderma harzianum on the reproduction of Meloidogyne incognita race 2 under greenhouse conditions

Interactive effects of alternatives to methyl bromide on root-knot (Meloidogyne species) nematodes could provide information on whether to use the products combined or singularly. The objective of this study was to investigate the interactive effects of Nemarioc-AL phytonematicide, Steinernema feltiae and Trichoderma harzianum on eggs and reproductive factors of Meloidogyne incognita race 2 under greenhouse conditions. The interactive effects of Nemarioc-AL phytonematicide (N), S. feltiae (S) and T. harzianum (T) on population densities of M. incognita were examined in a 2?×?2?×?2 factorial experiment in pots filled with steam-pasteurised soil. Each tomato seedling was inoculated with 2000 eggs and second-stage juveniles (J2), with phytonematicide and biocontrol agents applied at commercial rates. At 56 days after the treatments, the second-order (N₁S₁T₁) interaction reduced eggs and the reproductive factor (RF) in roots by 100% and 93%, respectively. Similarly, the first-order (N₁S₀T₁) interaction reduced the two variables by 92% and 72%, respectively, whereas N₁S₁T₀ interaction had 63% and 55% reduction effects, respectively. Nemarioc-AL phytonematicide was compatible with S. feltiae and T. harzianum and could, therefore, be used collectively to manage population densities of M. incognita.     

Monomeric L-amino acid oxidase-induced mitochondrial dysfunction in Rhizoctonia solani Reveals a novel antagonistic mechanism of Trichoderma harzianum ETS 323

The monomeric L-amino acid oxidase (mTh-LAAO) of Trichoderma harzianum ETS 323 has been suggested to antagonize Rhizoctonia solani by an unknown mechanism. Here, the mTh-LAAO-treated R. solani exhibited hyphal lysis and apoptotic characteristics such as DNA fragmentation, reactive oxygen species (ROS) accumulation, lipid peroxidation, and mitochondrial membrane potential depolarization. This hyphal lysis was suppressed by the mitochondria-dependent apoptosis inhibitor oligomycin while accompanied by reduction of ROS accumulation. This result suggested that mitochondria-mediated apoptosis in R. solani was involved in mTh-LAAO-induced growth inhibition, which was supported by the evidence of cytocheome c release and activation of caspases 9 and 3. Furthermore, the data indicated that the mTh-LAAO-induced fungal cell death was also closely interrelated with the interaction of mTh-LAAO with R. solani hyphal cell wall proteins. These results illuminate the biological function and mechanism underlying the antagonistic action of T. harzianum mTh-LAAO against fungal pathogens.     

Cloning of a novel L-amino acid oxidase from Trichoderma harzianum ETS 323 and bioactivity analysis of overexpressed L-amino acid oxidase

L-amino acid oxidases (L-AAOs) have been isolated from many organisms, such as snake, and are known to have antibacterial activity. To the best of the authors' knowledge, this is the first report of the cloning of cDNA encoding a novel Trichoderma harzianum ETS 323 L-amino acid oxidase (Th-L-AAO). The protein was overexpressed in Escherichia coli and purified to homogeneity. Comparisons of its deduced amino acid sequence with the sequence of other L-AAOs revealed the similarity to be between 9 and 24%. The molecular mass of the purified protein was 52 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme substrate specificity was highest for L-phenylalanine, and its optimal pH and temperature for activity were 7 and 40 °C, respectively; exogenous metal ions had no significant effect on activity. Circular dichroism spectroscopy indicated that the secondary structure of Th-L-AAO is composed of 17% α-helices, 28% β-sheets, and 55% random coils. The bacterially expressed Th-L-AAO also mediated antibacterial activity against both gram-positive and gram-negative food spoilage microorganisms. Furthermore, a three-dimensional protein structure was created to provide more information about the structural composition of Th-L-AAO, suggesting that the N-terminal sequence of Th-L-AAO may have contributed to the antibacterial activity of this protein.     

Genetic and phenotypic diversity and random association of DNA markers of isolates of the fungal plant pathogen Sclerotinia sclerotiorum from soil on a fine geographic scale

Sclerotia of the soil-borne plant pathogen Sclerotinia sclerotiorum were collected from 1 m² area of the top 1.27 cm layer of soil in an alfalfa field in southeastern Washington state of the US. Out of 272 sclerotia collected, 40 were randomly selected and analyzed for genetic diversity in terms of microsatellite loci, mycelial compatibility groups (MCGs) and phenotypic diversity using five phenotypic traits (fungicide sensitivity, oxalic acid production, growth rate, colony color and virulence). Sixteen microsatellite haplotypes and 15 MCGs were found among the 40 isolates. The isolates showed three colony colors (beige, dark and white) on Difco PDA and exhibited significant differences in growth rate, oxalic acid production, and sensitivity to three fungicides, benomyl, fluazinam and iprodione. However, these isolates did not show differences in their ability to colonize detached pea leaves. No apparent relationship among the neutral genetic markers and the phenotypic traits was detected. Two of the haplotypes accounted 40% of the isolates, suggesting isolates of these haplotypes might be better adapted to the environmental conditions in this alfalfa field. Several lines of evidence indicated high levels of genetic diversity and potential outcrossing within the population of S. sclerotiorum: 1) high likelihood of five genetic populations based on Bayesian probability and the presence of admixed isolates; 2) random association of alleles in every pair-wise linkage disequilibrium test among eight independent microsatellite loci; 3) discordances between microsatellite haplotypes and MCGs and 4) lack of correspondence among the genetic markers and phenotypic traits. Multilocus Index of Association test suggested that outcrossing occurs only within interbreeding subpopulations of S. sclerotiorum.