Sm2, a paralog of the Trichoderma cerato-platanin elicitor Sm1, is also highly important for plant protection conferred by the fungal-root interaction of Trichoderma with maize

Background

The proteins Sm1 and Sm2 from the biocontrol fungus Trichoderma virens belong to the cerato-platanin protein family. Members of this family are small, secreted proteins that are abundantly produced by filamentous fungi with all types of life-styles. Some species of the fungal genus Trichoderma are considered as biocontrol fungi because they are mycoparasites and are also able to directly interact with plants, thereby stimulating plant defense responses. It was previously shown that the cerato-platanin protein Sm1 from T. virens - and to a lesser extent its homologue Epl1 from Trichoderma atroviride - induce plant defense responses. The plant protection potential of other members of the cerato-platanin protein family in Trichoderma, however, has not yet been investigated.

Results

In order to analyze the function of the cerato-platanin protein Sm2, sm1 and sm2 knockout strains were generated and characterized. The effect of the lack of Sm1 and Sm2 in T. virens on inducing systemic resistance in maize seedlings, challenged with the plant pathogen Cochliobolus heterostrophus, was tested. These plant experiments were also performed with T. atroviride epl1 and epl2 knockout strains. In our plant-pathogen system T. virens was a more effective plant protectant than T. atroviride and the results with both Trichoderma species showed concordantly that the level of plant protection was more strongly reduced in plants treated with the sm2/epl2 knockout strains than with sm1/epl1 knockout strains.

Conclusions

Although the cerato-platanin genes sm1/epl1 are more abundantly expressed than sm2/epl2 during fungal growth, Sm2/Epl2 are, interestingly, more important than Sm1/Epl1 for the promotion of plant protection conferred by Trichoderma in the maize-C. heterostrophus pathosystem.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-014-0333-0) contains supplementary material, which is available to authorized users.     

Enzyme activity of extracellular protein induced in Trichoderma asperellum and T. longibrachiatum by substrates based on Agaricus bisporus and Phymatotrichopsis omnivora

Antagonistic Trichoderma spp. are used throughout the world for the biological control of soil-borne plant diseases. This approach has stimulated an on-going search for more efficient mycoparasitic strains with a high potential for producing extracellular lytic enzymes. This study compares the production of lytic enzymes by native strains of Trichoderma asperellum and Trichoderma longibrachiatum on substrates of differing complexity. The quantity of protein induced by Agaricus bisporus-based medium was higher than that induced by Phymatotrichopsis omnivora-based medium. In P. omnivora medium, T. asperellum exhibited higher chitinolytic and β-1,3-glucanolytic activities than T. longibrachiatum. The enzyme profile was related to the previously reported ability of these strains to inhibit the growth of several soil-borne plant pathogens. NAGase production was similar among the tested indigenous strains of T. longibrachiatum; T479 and T359 produced more endochitinase, T479 produced more glucanase, and T341 and T359 produced more β-1,3-glucanase. The detected variations in glucanase and β-1,3-glucanase activities suggest that the production of these enzymes is strongly influenced by the substrate. Strains T397 and T359 exhibited xylanase activity, which triggers defence mechanisms in plants. Thus, these strains may utilise an additional mechanism of biocontrol.     

The role of the high potential form of the cytochrome b559: Study of Thermosynechococcus elongatus mutants

Cytochrome b559 is an essential component of the photosystem II reaction center in photosynthetic oxygen-evolving organisms, but its function still remains unclear. The use of photosystem II preparations from Thermosynechococcus elongatus of high integrity and activity allowed us to measure for the first time the influence of cytochrome b559 mutations on its midpoint redox potential and on the reduction of the cytochrome b559 by the plastoquinone pool (or Q_B). In this work, five mutants having a mutation in the α-subunit (I14A, I14S, R18S, I27A and I27T) and one in the β-subunit (F32Y) of cytochrome b559 have been investigated. All the mutations led to a destabilization of the high potential form of the cytochrome b559. The midpoint redox potential of the high potential form was significantly altered in the αR18S and αI27T mutant strains. The αR18S strain also showed a high sensitivity to photoinhibitory illumination and an altered oxidase activity. This was suggested by measurements of light induced oxidation and dark re-reduction of the cytochrome b559 showing that under conditions of a non-functional water oxidation system, once the cytochrome is oxidized by P680⁺, the yield of its reduction by Q_B or the PQ pool was smaller and the kinetic slower in the αR18S mutant than in the wild-type strain. Thus, the extremely positive redox potential of the high potential form of cytochrome b559 could be necessary to ensure efficient oxidation of the PQ pool and to function as an electron reservoir replacing the water oxidation system when it is not operating.     

德国鸢尾对Cd胁迫的生理生态响应及积累特性

通过盆栽研究了Cd胁迫下德国鸢尾的生长状况、生态效应、生理特性及吸收和富集Cd的能力.结果表明:德国鸢尾对小于5 mg/kg的Cd有较强的耐性,适用于城区土壤修复;Cd浓度大于5 mg/kg时抑制德国鸢尾生长,降低了其生态效应.随着Cd浓度的增大,德国鸢尾根系活力、叶绿素含量和含水量逐渐降低,游离脯氨酸和可溶性糖含量先升高后降低,细胞膜透性逐渐升高.Cd在德国鸢尾体内分布为根系＞地上部分,随着Cd浓度的增大,德国鸢尾根系和地上部分Cd积累浓度逐渐升高、富集系数和转运系数逐渐降低;Cd浓度为20 mg/kg时德国鸢尾对Cd的积累量最大,为2.122 mg/plant.     

Trichoderma–Plant–Pathogen Interactions: Advances in Genetics of Biological Control

Trichoderma spp. are widely used in agriculture as biofungicides. Induction of plant defense and mycoparasitism (killing of one fungus by another) are considered to be the most important mechanisms of Trichoderma-mediated biological control. Understanding these mechanisms at the molecular level would help in developing strains with superior biocontrol properties. In this article, we review our current understanding of the genetics of interactions of Trichoderma with plants and plant pathogens.     

Optimization of potassium for proper growth and physiological response of Houttuynia cordata Thunb.

Houttuynia cordata Thunb. is an edible herb with a variety of pharmacological activities, but only limited information is available about its response towards potassium supplementation. Sterile plantlets were cultured in media with different potassium levels, and parameters related to growth, foliar potassium, water and chlorophyll contents, photosynthesis, transpiration, H₂O₂ contents and antioxidative enzyme activities were determined after a month. Results showed that 1.28 mM potassium was the optimum for H. cordata as highest values of dry weight, shoot height, root length and number were obtained at this concentration. The optimum potassium concentration resulted in the maximum net photosynthetic rate which could be associated with the highest chlorophyll content rather than limited stomatal conductance. The supply of surplus potassium resulted in higher content of foliar potassium, but negatively correlated with the biomass. Both potassium starvation (0 mM) and high potassium (>1.28 mM) could lead to water loss through high transpiration rate and low water absorption, respectively, and resulted in H₂O₂ accumulation and increased activities of catalase and peroxidase, which suggested induction of oxidative stress. Moreover, H. cordata showed the minimum of H₂O₂ content and the maximum of superoxide dismutase activity on 1.28 mM potassium, implying its role in inducing tolerance against oxidative stress.     

长苞香蒲对人工盐碱湿地Na+和K+的吸收与转运特征

为揭示长苞香蒲(Typha domingensis)对盐生湿地生态系统中Na⁺和K⁺的吸收与转运特征,探讨长苞香蒲对盐生湿地的生态修复效果,该研究采用人工模拟盐生湿地的方法,设置CK(对照)、T1(浇灌100 mmol·L^-1盐水)、T2(浇灌200 mmol·L^-1盐水)及T3(浇灌300 mmol·L^-1盐水)4种不同盐浓度的人工湿地生态系统,并分别于5月5日(开始盐胁迫处理,S0)、5月30日(S1)、6月30日(S2)和7月30日(S3)测量其株高和干重、植株地上与地下部分Na⁺和K⁺的含量以及底泥和水体中Na⁺和K⁺的含量以分析长苞香蒲对盐碱湿地的脱盐作用。结果表明：(1)各处理的长苞香蒲的株高和干重随着处理时间的延长呈增加趋势,但与CK相比,各处理生长量随盐浓度升高出现下降趋势。(2)高浓度盐处理(T3)使长苞香蒲的地上部分和地下部分的Na⁺分别增加了2.5...     

Water deficit in field-grown Gossypium hirsutum primarily limits net photosynthesis by decreasing stomatal conductance,increasing photorespiration,and increasing the ratio of dark respiration to gross photosynthesis

Much effort has been expended to improve irrigation efficiency and drought tolerance of agronomic crops; however, a clear understanding of the physiological mechanisms that interact to decrease source strength and drive yield loss has not been attained. To elucidate the underlying mechanisms contributing to inhibition of net carbon assimilation under drought stress, three cultivars of Gossypium hirsutum were grown in the field under contrasting irrigation regimes during the 2012 and 2013 growing season near Camilla, Georgia, USA. Physiological measurements were conducted on three sample dates during each growing season (providing a broad range of plant water status) and included, predawn and midday leaf water potential (Ψ_PD and Ψ_MD), gross and net photosynthesis, dark respiration, photorespiration, and chlorophyll a fluorescence. End-of-season lint yield was also determined. Ψ_PD ranged from −0.31 to −0.95 MPa, and Ψ_MD ranged from −1.02 to −2.67 MPa, depending upon irrigation regime and sample date. G. hirsutum responded to water deficit by decreasing stomatal conductance, increasing photorespiration, and increasing the ratio of dark respiration to gross photosynthesis, thereby limiting P_N and decreasing lint yield (lint yield declines observed during the 2012 growing season only). Conversely, even extreme water deficit, causing a 54% decline in P_N, did not negatively affect actual quantum yield, maximum quantum yield, or photosynthetic electron transport. It is concluded that P_N is primarily limited in drought-stressed G. hirsutum by decreased stomatal conductance, along with increases in respiratory and photorespiratory carbon losses, not inhibition or down-regulation of electron transport through photosystem II. It is further concluded that Ψ_PD is a reliable indicator of drought stress and the need for irrigation in field-grown cotton.     

The Influence of Trichoderma harzianum on the Root-knot Fusarium Wilt Complex in Cotton

Wilt-susceptible cultivar ''Rowden'' cotton was inoculated wilh Meloidogyne incognita (N), Trichoderma harzianum (T), and Fusarium oxysporum f. sp. vasinfectum (F) alone and in all combinations in various time sequences. Plants inoculated with F alone or in combination with T did not develop wilt, Simultaneous inoculation of 7-day-old seedlings with all three organisms (NTF) produced earliest wilt. However, plants receiving nematodes at 7 days and Fusarium and Trichoderma at 2 or 4 weeks later (N-T-F, N-TF) developed the greatest wilt between 49-84 days after initial nematode inoculation. During the same period, Fusarium added 4 weeks after initial nematode inoculation (N-F) and Fusarium added 4 weeks after initial simultaneous inoculation of nematode and Trichoderma (NT-F) produced the least wilt. The addition of Fusarium inhibited nematode reproduction. Simultaneous inoculation with nematodes and Trichoderma (NT-) resulted in the greatest root gall development, whereas nematodes alone produced the greatest number of larvae. In comparison with noninoculated controls (CK), treatments involving all three organisms inhibited plant growth, plants inoculated with the nematode alone (N-) or with nematodes and Trichoderma (NT-) simultaneously had greatest root weight. Any treatment involving the nematode resulted in fewer bolls per plant and greater necrosis on roots than the noninoculated checks.     

外源磷或有机质对板蓝根吸收转运砷的影响

采用土壤培养方法,研究了不同含砷水平土壤中添加外源磷或有机质对砷在板蓝根地下部和地上部累积与分配的影响。结果表明,在外源添加磷或者有机质的情况下,与自然土相比含砷土对板蓝根的生长有一定的促进作用;在自然土(13.4 mg/kg)中,外源磷没有明显影响板蓝根地下部对砷的累积,却显著降低了砷由地下部向地上部的转运,并且添加200 mg P2 O5/kg显著降低了砷在地上部的累积。然而,在含砷土(33.4 mg/kg)中,100 mg P2O5/kg处理显著降低了砷在地下部的累积,但随磷用量的增加反而促进了地下部砷的累积;在添加有机质试验中,10 g/kg的有机质显著降低了自然土中板蓝根地下部和地上部对砷的累积,并且砷的吸收能力也明显下降。在含砷土(23.4 mg/kg)中,添加5 g/kg的有机质不仅降低了砷在板蓝根中的富集,而且降低了其对砷的吸收能力,提高了砷由地下部向地上部的转运,但是随着有机质施用量增至10 g/kg,地下部砷含量及其吸收砷的能力均有一定程度的增大。因此,在砷水平较低的自然土壤上种植板蓝根添加200 mg P2O5/kg和10 g/kg的有机质是控制砷在该草药体内积累的适宜用量,而在砷水平较高的土壤上100 mg P2O5/kg和5 g/kg的有机质是降低板蓝根体内砷累积的适宜用量。     

Detection of Mycoplasma mycoides subsp. mycoides SC in bronchoalveolar lavage fluids of cows based on a TaqMan real-time PCR discriminating wild type strains from an lppQ mutant vaccine strain used for DIVA-strategies

Contagious bovine pleuropneumonia (CBPP) is the most serious cattle disease in Africa, caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC). CBPP control strategies currently rely on vaccination with a vaccine based on live attenuated strains of the organism. Recently, an lppQ⁻ mutant of the existing vaccine strain T1/44 has been developed (Janis et al., 2008). This T1lppQ⁻ mutant strain is devoid of lipoprotein LppQ, a potential virulence attribute of M. mycoides subsp. mycoides SC. It is designated as a potential live DIVA (Differentiating Infected from Vaccinated Animals) vaccine strain allowing both serological and etiological differentiation. The present paper reports on the validation of a control strategy for CBPP in cattle, whereby a TaqMan real-time PCR based on the lppQ gene has been developed for the direct detection of M. mycoides subsp. mycoides SC in ex vivo bronchoalveolar lavage fluids of cows and for the discrimination of wild type strains from the lppQ⁻ mutant vaccine strain.     

Water status in Mesembryanthemum crystallinum under heavy metal stress

Heavy metals (HMs) are known to have negative effects on plant water status; however, the mechanisms by which plants rearrange their water relations to adapt to such conditions are poorly understood. Using the model plant Mesembryanthemum crystallinum, we studied disturbances in water status and rapid plant defence responses induced by excess copper or zinc. After a day of HM stress, reductions in root sap exudation and water deficits in leaf tissues became evident. We also observed several primary adaptive events, including a rapid decrease in the transpiration rate and progressive declines in the leaf-cell sap osmotic potential. Longer HM treatments resulted in reductions of total and relative water contents as well as proline accumulation, an increase in water retention capacity and changes in aquaporin gene expression. After 3 h of HM exposure, leaf expression of the McTIP2;2 gene, which encodes tonoplast aquaporin, was suppressed more than two-fold, thus representing one of the earliest responses to HM treatment. The expression of three additional aquaporin genes was also reduced starting at 9 h; this effect became more prominent upon longer HM exposure. These results indicate that HMs induce critical rearrangements in the water relations of M. crystallinum plants, based on the rapid suppression of transpiration flow and strong inhibition of root sap exudation. These effects then triggered an adaptive water-conserving strategy involving differential regulation of aquaporin gene expression in leaves and roots, further reductions in transpiration, and an accelerated switch to CAM photosynthesis.     

Definition of two new symbiovars,sv. lupini and sv. mediterranense, within the genera Bradyrhizobium and Phyllobacterium efficiently nodulating Lupinus micranthus in Tunisia

In this study, a polyphasic approach was used to analyze three representative strains (LmiH4, LmiM2 and LmiT21) from a collection of six previously described strains isolated in Tunisia from root nodules of Lupinus micranthus. The phylogenetic analysis of the concatenated rrs, recA and glnII genes showed that strain LmiH4 had 100% concatenated gene sequence identity with the type strain Bradyrhizobium retamae Ro19T. Similarly, strain LmiM2 shared 100% concatenated gene sequence identity with the species Bradyrhizobium valentinum LmjM3T. However, strain LmiT21 showed an identical concatenated gene sequence with reference strain Phyllobacterium sophorae CCBAU03422T. The recA-glnII concatenated protein-coding genes used produced incongruent phylogenies compared with 16S rDNA phylogeny. The nodC gene analysis showed that the strains were phylogenetically divergent to the Bradyrhizobium symbiovars defined to date, and represented two new symbiovars. Plant infection analysis revealed that the three strains showed moderate host range and symbiotic specificities.Based on their symbiotic characteristics, we propose that the three strains isolated from Lupinus micranthus nodules belong to two new symbiovars, with the first denominated lupini within the two species Bradyrhizobium valentinum (type strain LmiM2) and B. retamae (type strain LmiH4), and the second denominated mediterranense within the species P. sophorae (type strain LmiT21).     

A novel insight into the cost–benefit model for the evolution of botanical carnivory

Background The cost–benefit model for the evolution of botanical carnivory provides a conceptual framework for interpreting a wide range of comparative and experimental studies on carnivorous plants. This model assumes that the modified leaves called traps represent a significant cost for the plant, and this cost is outweighed by the benefits from increased nutrient uptake from prey, in terms of enhancing the rate of photosynthesis per unit leaf mass or area (A_N) in the microsites inhabited by carnivorous plants.Scope This review summarizes results from the classical interpretation of the cost–benefit model for evolution of botanical carnivory and highlights the costs and benefits of active trapping mechanisms, including water pumping, electrical signalling and accumulation of jasmonates. Novel alternative sequestration strategies (utilization of leaf litter and faeces) in carnivorous plants are also discussed in the context of the cost–benefit model.Conclusions Traps of carnivorous plants have lower A_N than leaves, and the leaves have higher A_N after feeding. Prey digestion, water pumping and electrical signalling represent a significant carbon cost (as an increased rate of respiration, R_D) for carnivorous plants. On the other hand, jasmonate accumulation during the digestive period and reprogramming of gene expression from growth and photosynthesis to prey digestion optimizes enzyme production in comparison with constitutive secretion. This inducibility may have evolved as a cost-saving strategy beneficial for carnivorous plants. The similarities between plant defence mechanisms and botanical carnivory are highlighted.     

Overexpression of erg20 gene encoding farnesyl pyrophosphate synthase has contrasting effects on activity of enzymes of the dolichyl and sterol branches of mevalonate pathway in Trichoderma reesei

The mevalonate pathway is the most diverse metabolic route resulting in the biosynthesis of at least 30,000 isoprenoid compounds, many of which, such as sterols or dolichols, are indispensable for living cells. In the filamentous fungus Trichoderma of major biotechnological interest isoprenoid metabolites are also involved in the biocontrol processes giving the mevalonate pathway an additional significance. On the other hand, little is known about genes coding for enzymes of the mevalonate pathway in Trichoderma.     

Tylenchulus semipenetrans Alters the Microbial Community in the Citrus Rhizosphere

Infection of citrus seedlings by Tylenchulus semipenetrans was shown to reduce subsequent infection of roots by Phytophthora nicotianae and to increase plant growth compared to plants infected by only the fungus. Hypothetical mechanisms by which the nematode suppresses fungal development include nutrient competition, direct antibiosis, or alteration of the microbial community in the rhizosphere to favor microorganisms antagonistic to P. nicotianae. A test of the last hypothesis was conducted via surveys of five sites in each of three citrus orchards infested with both organisms. A total of 180 2-cm-long fibrous root segments, half with a female T. semipenetrans egg mass on the root surface and half without, were obtained from each orchard site. The samples were macerated in water, and fungi and bacteria in the suspensions were isolated, quantified, and identified. No differences were detected in the numbers of microorganism species isolated from nematode-infected and uninfected root segments. However, nematode-infected root segments had significantly more propagules of bacteria at all orchard sites. Bacillus megaterium and Burkholderia cepacia were the dominant bacterial species recovered. Bacteria belonging to the genera Arthrobacter and Stenotrophomonas were encountered less frequently. The fungus community was dominated by Fusarium solani, but Trichoderma, Verticillum, Phythophthora, and Penicillium spp. also were recovered. All isolated bacteria equally inhibited the growth of P. nicotianae in vitro. Experiments using selected bacteria, T. semipenetrans, and P. nicotianae, alone or in combination, were conducted in both the laboratory and greenhouse. Root and stem fresh weights of P. nicotianae-infected plants treated with T. semipenetrans, B. cepacia, or B. megaterium were greater than for plants treated only with the fungus. Phytophthora nicotianae protein in roots of fungus-infected plants was reduced by nematodes (P ≤ 0.001), either alone or in combination with either bacterium. However, treatment with bacteria did not affect P. nicotianae development in roots. The results suggest different mechanisms by which T. semipenetrans, B. cepacia, and B. megaterium may mitigate virulence of P. nicotianae.     

Inoculation of Rhizobium (VR-1 and VA-1) induces an increasing growth and metal accumulation potential in Vigna radiata and Vigna angularis L. growing under fly-ash

Fly-ash-tolerant Rhizobium strains were isolated from plants grown in fly-ash-contaminated soil, axenically under laboratory conditions. Saplings of both plants were raised in N₂-free Jenson medium and inoculated with 2.6 × 10⁸ cell ml⁻¹ and 5.2 × 10⁸ cell ml⁻¹ of culture after 10 d of growth. Plants were transferred into 100% fly-ash under natural condition. Rhizobium-inoculated plants grown on 100% fly-ash showed marked increase in relation to root-shoot length, biomass yield, photosynthetic pigment, protein content and nodulation frequency compared to uninoculated plant grown in control (100% fly-ash). Inoculation of fly-ash-tolerant Rhizobium increased the accumulation of Fe, Zn, Cu Cd and Cr in different tissues vis-à-vis enhanced translocation of metals to the aboveground part of plant. Although inoculation of fly-ash-tolerant Rhizobium strains (VR-1 and VA-1) enhanced the translocation of more Fe to shoot parts, nevertheless, the amount of Rhizobium inoculants supplied to the plant was found to be very important since it has a positive role in increasing plant growth through increased N₂ supply via nitrogenase activity. Results suggest that an integrated approach employing biotechnological means and inoculation of plants with host-specific fly-ash-tolerant Rhizobium strain may prove a stimulus to a fly-ash management programme.     

Study of different Trichoderma strains on growth characteristics and silymarin accumulation of milk thistle plant

Abstract

Biological control involves the use of beneficial organisms such as Trichoderma that promote positive responses by the plant. Trichoderma species produce and/or release a variety of compounds, including cell wall degrading enzymes and secondary metabolites, which enhance root development, crop productivity and resistance to biotic and abiotic stresses. This study examines the effects of the five Trichoderma strains (M7, KHB, G124-1, G46-3 and G46-7) on milk thistle including morphological characteristics and silymarin accumulation. This research indicated that treating milk thistle by M7 strain, significantly increased the highest branch length of plants, while strain KHB induced the production of silychristin, isosilybin and silymarin. Finally, strain G46-7 dramatically increased silydianin content in milk thistle plant. The findings suggested that certain Trichoderma strains are positive elicitors for promoting growth characteristics and silymarin accumulation in Silybum marianum (L.) Gaertn.     

Volatile organic compounds of some Trichoderma spp. increase growth and induce salt tolerance in Arabidopsis thaliana

Many beneficial effects of Trichoderma spp. on plant growth and/or resistance to biotic/abiotic stresses can result from the production of bioactive compounds including volatile organic compounds (VOCs). We evaluated the effects of the volatile mixtures from 13 strains of different Trichoderma species on induction of tolerance to salt stress (100 mM NaCl) as well as growth promotion of Arabidopsis thaliana. Plants responded differently due to the presence of VOCs from various Trichoderma species ranging from both growth promotion and induction of salt tolerance to no significant changes under any of the conditions tested. In plants exposed for 2 weeks to VOCs of the selected strain, i.e. Trichoderma koningii, there was less H₂O₂ accumulation under salt stress compared to that in control plants. This result may reflect the possible role of VOCs of this strain in plant protection against oxidative damage under salt stress. Together, induction of salt tolerance using VOCs should be added to the known mechanisms of plant vigor enhancement by Trichoderma spp.