Isolation of two aspartyl proteases from Trichoderma asperellum expressed during colonization of cucumber roots

Trichoderma asperellum and cucumber seedlings were used as a model to study the modulation of Trichoderma gene expression during plant root colonization. Seedlings were grown in an aseptic hydroponics medium and inoculated with Trichoderma spore suspension. Proteins differentially secreted into the medium were isolated. Three major proteins of fungal origin were identified: two arabinofuranosidases (Abf1 and Abf2) and an aspartyl protease. Differential mRNA display was conducted on Trichoderma mycelia interacting and non-interacting, with the plant roots. Among the differentially regulated clones another aspartyl protease was identified. Sequencing of the genes revealed that the first aspartyl protease is a close homologue of PapA from T. harzianum and the other, of AP1 from Botryotinia fuckeliana. RT-PCR analysis confirms that the proteases are induced in response to plant roots attachment and are expressed in planta. papA, but not papB, is also induced in plate confrontation assays with the plant pathogen Rhizoctonia solani. These data suggest that the identified proteases play a role in Trichoderma both as a mycoparasite and as a plant opportunistic symbiont.     

Proteome, salicylic acid, and jasmonic acid changes in cucumber plants inoculated with Trichoderma asperellum strain T34

Trichoderma spp. is one of the most commonly used biological control agents against plant pathogens. This fungus produces changes in plant metabolism, thus increasing growth and enhancing resistance to biotic and abiotic stresses. However, its modes of action remain to be defined. In the first hours of interaction between cucumber plant roots and Trichoderma asperellum strain T34, salicylic and jasmonic acid levels and typical antipathogenic peroxidase activity increase in the cotyledons to different degrees depending on the applied concentration of the fungi. The use of 2-DE protein profiling and MS analysis allowed us to identify 28 proteins whose expression was affected in cotyledons after cucumber root colonization by Trichoderma applied at high concentrations: 17 were found to be up-regulated while 11 were down-regulated. Proteins involved in ROS scavenging, stress response, isoprenoid and ethylene biosynthesis, and in photosynthesis, photorespiration, and carbohydrate metabolism were differentially regulated by Trichoderma. The proteome changes found in this study help to give an understanding of how Trichoderma-treated plants become more resistant to pathogen attacks through the changes in expression of a set of defence-oriented proteins which can directly protect the plant or switch the metabolism to a defensive, nonassimilatory state.     

Use of GUS Transformants of Trichoderma harzianum Isolate T39 (TRICHODEX) for Studying Interactions on Leaf Surfaces

Trichoderma harzianum , isolate T39 (TRICHODEX), was transformed with the GUS ( βglucuronidase) reporter gene to visualize the interaction with Botrytis cinerea and to determine potential mechanisms involved in biocontrol. T39GUS transformants did not appear to penetrate the bean leaf epidermis nor grow into the mesophyll via stomata. No direct penetration or coiling was observed between hyphae and conidia of the T39GUS transformants and of B. cinerea , however, structures of the pathogen were stained blue. Similarly, blue substrate accumulated in various non-transformed fungi such as Alternaria sp., Colletotricum acutatum and the wild-type isolate T39 when co-inoculated on leaves and plates with the T39GUStransgenic strains of T. harzianum and Fusarium oxysporum f. sp. niveum . Therefore, the role of diffusible compounds, specifically from T. harzianum transformants, will need further investigation.     

花青素对神经退行性疾病保护作用的研究进展

对近年来花青素在神经退行性疾病中的研究进展进行综述,并从抑制氧化应激、缓解神经炎症、缓解兴奋性毒性和抑制蛋白异常聚集等方面对花青素的神经保护作用机制进行探讨,为进一步了解和研究花青素类化合物在神经退行性疾病中的预防作用提供科学依据。     

Chronological Events Associated with the Antagonistic Properties of Trichoderma harzianum against Botrytis cinerea: Indirect Evidence for Sequential Role of Antibiosis and Parasitism

Chronological events associated with the degradation of Botrytis cinerea by a strain of Trichoderma harzianum selected for superior biocontrol ability were studied using ultrastructure and cytochemical investigations in an attempt to define the relative roles of antibiosis and parasitism in the antagonistic process. The first ultrastructural changes were observed 12 h before contact between the organisms, and were characterized by punctuated invaginations of the Botrytis plasmalemma. These reactions were followed by a gradual retraction of the plasmalemma, disorganization of the cytoplasm, loss of turgor pressure and cell death within 48 h of contact between hyphae of the interacting fungi. The first evidence of penetration of B. cinerea by T. harzianum was recorded 72 h after contact. This penetration was apparently mediated by either mechanical pressure or localized wall digestion at points of entry, as there was no clear evidence of chitinolytic degradation of the B. cinerea cell wall, as determined by cytochemical labelling of chitin with a lectin-gold conjugate. However, after 10 days there was clear indication of chitin degradation, based on the random and reduced presence of gold particles over the cell wall of B. cinerea. These results suggest that the strain of T. harzianum antagonized first and foremost by antibiosis, leading to cell death, followed by degradation of the cell by means of chitinolytic enzymes. The production of antibiotics may, therefore, be more important than that of chitinolytic enzymes in conferring superior biocontrol properties to T. harzianum.     

Biomimics of fungal cell-cell recognition by use of lectin-coated nylon fibers.

When the mycoparasitic, biocontrol fungus Trichoderma harzianum was allowed to grow on nylon fibers treated with concanavalin A or Sclerotium rolfsii lectin, it coiled around the nylon fibers and produced hooks in a pattern similar to that observed with the real host hyphae. The incidence of interaction between T. harzianum and S. rolfsii lectin-treated fibers was significantly higher than that of the controls (untreated or blocked activated fibers). These findings provide direct evidence for the role of lectins in mycoparasitism.     

木霉对辣椒疫霉菌抑制作用的超微结构与细胞化学

哈茨木霉菌株NF9和TC3对辣椒疫霉病菌具有强烈的抑制作用。超微结构与细胞化学研究表明,木霉菌丝能够缠绕并寄生疫霉菌菌丝,且重寄生作用主要发生在培养基内的菌丝上。但在绝大多数情况下,木霉菌可直接水解和破坏疫霉菌菌丝,说明木霉抑菌作用的主要机制是直接向外分泌水解酶分解疫霉的细胞壁及细胞质,而不是重寄生作用。不同培养基对木霉的重寄生作用有重要影响,减少培养基中葡萄糖含量可以增强木霉的重寄生作用和抑菌效果。此外,在菌落生长后期,疫霉的新生菌丝均可侵染自身的老菌丝。     

Cellular and Molecular Mechanisms Involved in the Interaction between Trichoderma harzianum and Pythium ultimum

The interaction between Trichoderma harzianum and the soilborne plant pathogen Pythium ultimum was studied by electron microscopy and further investigated by gold cytochemistry. Early contact between the two fungi was accompanied by the abnormal deposition of a cellulose-enriched material at sites of potential antagonist penetration. The antagonist displayed the ability to penetrate this barrier, indicating that cellulolytic enzymes were produced. However, the presence of cellulose in the walls of severely damaged Pythium hyphae indicated that cellulolytic enzymes were not the only critical traits involved in the antagonistic process. The marked alteration of the (beta)-1,3-glucan component of the Pythium cell wall suggested that (beta)-1,3-glucanases played a key role in the process.     

Interactions between Glomus mosseae and arbuscular mycorrhizal sporocarp-associated saprophytic fungi

The saprophytic fungi Wardomyces inflatus (Marchal) Hennebert, Paecilomyces farinosus (Holm & Gray) A. H. S. Brown & G. Sm., Gliocladium roseum Bain., sterile dark mycelium (SDM-54), Trichoderma pseudokoningii Rifai and Trichoderma harzianum Rifai were isolated from sporocarps of Glomus mosseae. The effect of saprophytic fungi on G. mosseae spore germination was tested on water agar. Wardomyces inflatus decreased the percent germination of G. mosseae spores; G. roseum, T. pseudokoningii and T. harzianum had no effect on germination; and P. farinosus and SDM-54 increased the percentage of spore germination of G. mosseae after 4 d. Wardomyces inflatus significantly decreased hyphal length of spores which germinated, but no other saprophytic fungi affected hyphal growth. Trichoderma pseudokoningii, T. harzianum, P. farinosus and SDM-54 increased the number of auxiliary cells formed by G. mosseae. The effect of saprophytic fungi on arbuscular mycorrhizal colonization of soybean was studied in a greenhouse trial. The percentage of soybean root length colonized was decreased by W. inflatus, unaffected by SDM-54 and T. harzianum, and increased by P. farinosus. Gliocladium roseum decreased root length colonized when plants were 12 wk old, and T. pseudokoningii increased colonization of roots when plants were 4 wk old. Antagonistic, synergistic and neutral actions of G. mosseae upon the saprophytic fungi were observed. The population of T. harzianum decreased and the populations of T. pseudokoningii and SDM-54 increased in the presence of G. mosseae. Our results indicate a complex interaction between G. mosseae and associated saprophytic fungi.     

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.     

Common mycorrhizal networks provide a potential pathway for the transfer of hydraulically lifted water between plants

Plant roots may be linked by shared or common mycorrhizal networks (CMNs) that constitute pathways for the transfer of resources among plants. The potential for water transfer by such networks was examined by manipulating CMNs independently of plant roots in order to isolate the role(s) of ectomycorrhizal (EM) and arbuscular mycorrhizal fungal (AMF) networks in the plant water balance during drought (soil water potential -5.9 MPa). Fluorescent tracer dyes and deuterium-enriched water were used to follow the pathways of water transfer from coastal live oak seedlings (Quercus agrifolia Nee; colonized by EM and AMF) conducting hydraulic lift (HL) into the roots of water-stressed seedlings connected only by EM (Q. agrifolia) or AMF networks (Q. agrifolia, Eriogonum fasciculatum Benth., Salvia mellifera Greene, Keckiella antirrhinoides Benth). When connected to donor plants by hyphal linkages, deuterium was detected in the transpiration flux of receiver oak plants, and dye-labelled extraradical hyphae, rhizomorphs, mantles, and Hartig nets were observed in receiver EM oak roots, and in AMF hyphae of Salvia. Hyphal labelling was scarce in Eriogonum and Keckiella since these species are less dependent on AMF. The observed patterns of dye distribution also indicated that only a small percentage of mycorrhizal roots and extraradical hyphae were involved with water transfer among plants. Our results suggest that the movement of water by CMNs is potentially important to plant survival during drought, and that the functional ecophysiological traits of individual mycorrhizal fungi may be a component of this mechanism.     

The molecular basis of shoot responses of maize seedlings to Trichoderma harzianum T22 inoculation of the root: a proteomic approach

Trichoderma spp. are effective biocontrol agents for several soil-borne plant pathogens, and some are also known for their abilities to enhance systemic resistance to plant diseases and overall plant growth. Root colonization with Trichoderma harzianum Rifai strain 22 (T22) induces large changes in the proteome of shoots of maize (Zea mays) seedlings, even though T22 is present only on roots. We chose a proteomic approach to analyze those changes and identify pathways and genes that are involved in these processes. We used two-dimensional gel electrophoresis to identify proteins that are differentially expressed in response to colonization of maize plants with T22. Up- or down-regulated spots were subjected to tryptic digestion followed by identification using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry and nanospray ion-trap tandem mass spectrometry. We identified 91 out of 114 up-regulated and 30 out of 50 down-regulated proteins in the shoots. Classification of these revealed that a large portion of the up-regulated proteins are involved in carbohydrate metabolism and some were photosynthesis or stress related. Increased photosynthesis should have resulted in increased starch accumulation in seedlings and did indeed occur. In addition, numerous proteins induced in response to Trichoderma were those involved in stress and defense responses. Other processes that were up-regulated were amino acid metabolism, cell wall metabolism, and genetic information processing. Conversely, while the proteins involved in the pathways noted above were generally up-regulated, proteins involved in other processes such as secondary metabolism and protein biosynthesis were generally not affected. Up-regulation of carbohydrate metabolism and resistance responses may correspond to the enhanced growth response and induced resistance, respectively, conferred by the Trichoderma inoculation.     

Trichoderma species form endophytic associations within Theobroma cacao trichomes

Trichoderma species are usually considered soil organisms that colonize plant roots, sometimes forming a symbiotic relationship. Recent studies demonstrate that Trichoderma species are also capable of colonizing the above ground tissues of Theobroma cacao (cacao) in what has been characterized as an endophytic relationship. Trichoderma species can be re-isolated from surface sterilized cacao stem tissue, including the bark and xylem, the apical meristem, and to a lesser degree from leaves. SEM analysis of cacao stems colonized by strains of four Trichoderma species (Trichoderma ovalisporum-DIS 70a, Trichoderma hamatum-DIS 219b, Trichoderma koningiopsis-DIS 172ai, or Trichoderma harzianum-DIS 219f) showed a preference for surface colonization of glandular trichomes versus non-glandular trichomes. The Trichoderma strains colonized the glandular trichome tips and formed swellings resembling appresoria. Hyphae were observed emerging from the glandular trichomes on surface sterilized stems from cacao seedlings that had been inoculated with each of the four Trichoderma strains. Fungal hyphae were observed under the microscope emerging from the trichomes as soon as 6 h after their isolation from surface sterilized cacao seedling stems. Hyphae were also observed, in some cases, emerging from stalk cells opposite the trichome head. Repeated single trichome/hyphae isolations verified that the emerging hyphae were the Trichoderma strains with which the cacao seedlings had been inoculated. Strains of four Trichoderma species were able to enter glandular trichomes during the colonization of cacao stems where they survived surface sterilization and could be re-isolated. The penetration of cacao trichomes may provide the entry point for Trichoderma species into the cacao stem allowing systemic colonization of this tissue.     

Interaction between Laccaria laccata and Trichoderma virens in co-culture and in the rhizosphere of Pinus sylvestris grown in vitro

Interactions between the ectomycorrhizal fungus Laccaria laccata and the soil fungus Trichoderma virens in co-culture and in the rhizosphere of Pinus sylvestris seedlings growing in vitro were investigated by light and scanning electron microscopy. The growth of T. virens was inhibited in co-culture. Shortened, more branched and sometimes deformed or injured hyphae of T. virens were observed in the zone of inhibition. Two-month-old mycorrhizae of P. sylvestris/ L. laccata were inoculated with a conidial suspension of T. virens and examined at intervals of 7-24 h and 2, 3, and 6 days post-inoculation (p.i.). On non-mycorrhizal roots, conidia germination was high and long hyphae formed 3 days p.i. On mycorrhizal roots, short germ tubes were observed only sporadically. At 3 days p.i., the mantle hyphae of L. laccata grew towards the conidia and coiled around them. Extremely dense coils of hyphae were found around clusters of conidia. Deformation of conidia, breaks in conidial walls and their partial degradation were observed 6 days p.i.     

Marine isolates of Trichoderma spp. as potential halotolerant agents of biological control for arid-zone agriculture

The scarcity of fresh water in the Mediterranean region necessitates the search for halotolerant agents of biological control of plant diseases that can be applied in arid-zone agriculture irrigated with saline water. Among 29 Trichoderma strains previously isolated from Mediterranean Psammocinia sp. sponges, the greatest number of isolates belong to the Trichoderma longibrachiatum-Hypocrea orientalis species pair (9), H. atroviridis/T. atroviride (9), and T. harzianum species complex (7), all of which are known for high mycoparasitic potential. In addition, one isolate of T. asperelloides and two putative new species, Trichoderma sp. O.Y. 14707 and O.Y. 2407, from Longibrachiatum and Strictipilosa clades, respectively, have been identified. In vitro salinity assays showed that the ability to tolerate increasing osmotic pressure (halotolerance) is a strain- or clade-specific property rather than a feature of a species. Only a few isolates were found to be sensitive to increased salinity, while others either were halotolerant or even demonstrated improved growth in increasingly saline conditions. In vitro antibiosis assays revealed strong antagonistic activity toward phytopathogens due to the production of both soluble and volatile metabolites. Two marine-derived Trichoderma isolates, identified as T. atroviride and T. asperelloides, respectively, effectively reduced Rhizoctonia solani damping-off disease on beans and also induced defense responses in cucumber seedlings against Pseudomonas syringae pv. lachrimans. This is the first inclusive evaluation of marine fungi as potential biocontrol agents.     

Variations in the level of enzyme activity and immunolocalization of calcium-dependent protein kinases in the phloem of different cucumber organs

Calcium-dependent protein kinases (CDPKs) constitute a unique family of enzymes in plants that are characterized by a C-terminal calmodulin (CaM)-like domain. Through protein kinase assays, we have examined the levels of cucumber calcium-dependent kinase (CsCDPK) activity in various organs of cucumber seedlings and plants. The activity of CsCDPK was highest in cucumber plant leaves followed by seedling roots and hypocotyls; however, cucumber plant flowers, seedling cotyledons, and hooks had levels that were barely detectable. The CsCDPKs were immunolocalized using polyclonal antibodies that are highly specific against a part of the kinase domain of a calcium-dependent protein kinase (CsCDPKS) in the phloem sieve elements (SEs) in various organs of cucumber. In addition, this study indicates the presence of CsCDPKs in organelle-like bodies associated with the plasma membrane of sieve elements in mature stems and roots as well as in the storage bodies of immature seeds. These findings are discussed in terms of the likely roles played by CDPKs in the signal transduction pathways for Ca2+-regulated phloem transport of assimilates from leaves to various organs during growth and development of cucumber seedlings and plants.     

Inhibition of flowering of cucumber grafted on rooted squash stock

For the elucidation of the mechanisms of floral transition in indifferent plants, cucumber seedlings ( Cucumis sativus cv. Rennsei or cv. Shimoshirazu-jibai) were grafted onto squash seedlings ( Cucurbita maxima Duchesne X C. moschata Duchesne cv. Shintosa-ichigou) of which the meristems had been removed, and the effect on flower induction on the cucumber scion was examined. In both cultivars, the grafted cucumber bore no flowers, whereas control plants developed flowers above the second to fourth nodes. The inhibition of flower formation on the grafted cucumber scion occurred even when the root of cucumber was left with the squash root on the grafted plant, and flower formation occurred after removal of the squash stock. The inhibitory effect of the squash stock in the presence of the cucumber root was abolished by removal of the squash root. Neither the dry weight of stem plus leaf nor the chlorophyll content of the leaf, as indicators of vegetative growth, were correlated with flower formation on cucumber plants that had been grafted in the presence of cucumber roots on whole, cotyledon-free or root-free squash stock. These results indicate that flower formation in cucumber was inhibited by a factor produced by squash roots, an inhibition probably not involved in the modulation of vegetative growth. The root may control floral transition by the production of inhibitory factors in some day-neutral Cucurbitaceae plants.     

Biological control of Sclerotinia sclerotiorum attacking soybean plants. Degradation of the cell walls of this pathogen by Trichoderma harzianum (BAFC 742)

Two experiments of biological control of Sclerotinia sclerotiorum, one in the greenhouse and the other in the field, were carried out with soybean and Trichoderma harzianum as host and antagonist, respectively. Significant control of disease was achieved in both experiments, but there were no significant differences in plant growths. In the greenhouse, the application of T. harzianum as alginate capsules, increased the survival of soybean plants more than 100% with respect to the disease treatment. In the field, T. harzianum treated plants survived 40% more than those from the disease treatment, showing a similar survival level to control plants. Besides, a significant reduction (62.5%) in the number of germinated sclerotia was observed in the Trichoderma treated plot. Chitinase and 1,3-β- glucanase activities were detected when T. harzianum was grown in a medium containing Sclerotinia sclerotiorum cell walls as sole carbon source. In addition, electrophoretic profiles of proteins induced in T. harzianum showed quantitative differences between major bands obtained in the media induced by S. sclerotiorum cell walls and that containing glucose as a sole carbon source. This revised version was published online in June 2006 with corrections to the Cover Date.