Diversity and heavy metal tolerance of endophytic fungi from Dysphania ambrosioides,a hyperaccumulator from Pb–Zn contaminated soils

The diversity and heavy metal (HM) tolerance of endophytic fungi (EF) associated with Dysphania ambrosioides, a hyperaccumulator from two Pb–Zn contaminated sites were investigated. A total of 237 culturable EF were isolated and identified to 43 taxa based on morphological characteristics and rDNA internal transcribed spacer analysis, of which 13 occurred as endophytes of both sites, while other taxa were only found in either site. The colonization rate, dominant genera, community structure of EF as well as the HM content in the plant from two sites were significantly different. We suggest that these differences may result from the difference in the soil HM content: lower HM content in the soil, more EF in the plant, which may enhance the plant HM accumulation and thus result higher HM in it. HM tolerance tests indicated that 50% of the isolates exhibited HM tolerance. Among them, two isolates exhibited better HM tolerance, of which FT2G59 could tolerate Pb, Zn, and Cd, and the minimum inhibitory concentration (MIC) of them were 30–50, >?680, 20–30?mmol/l, respectively. While, the isolate FT2G7 could tolerate Cd, and the MIC was 30–50?mmol/l. These isolates may have potential application in phytoremediation.     

ENDOPHYTIC BACTERIA FROM SEEDS OF NICOTIANA TABACUM CAN REDUCE CADMIUM PHYTOTOXICITY

Although endophytic bacteria seem to have a close association with their host plant, little is known about the influence of seed endophytic bacteria on initial plant development and on their interactions with plants under conditions of metal toxicity. In order to further elucidate this close relationship, we isolated endophytic bacteria from surface sterilized Nicotiana tabacum seeds that were collected from plants cultivated on a cadmium-(Cd) and zinc-enriched soil. Many of the isolated strains showed Cd tolerance. Sterilely grown tobacco plants were inoculated with either the endogenous microbial consortium, composed of cultivable and noncultivable strains; single strains; or defined consortia of the most representative cultivable strains. Subsequently, the effects of inoculation of endophytic bacteria on plant development and on metal and nutrient uptake were explored under conditions with and without exposure to Cd. In general, seed endophytes were found to have a positive effect on plant growth, as was illustrated by an increase in biomass production under conditions without Cd. In several cases, inoculation with endophytes resulted in improved biomass production under conditions of Cd stress, as well as in a higher plant Cd concentration and total plant Cd content compared to noninoculated plants. These results demonstrate the beneficial effects of seed endophytes on metal toxicity and accumulation, and suggest practical applications using inoculated seeds as a vector for plant beneficial bacteria.     

重金属胁迫下内生菌对宿主植物的解毒机制

采用内生菌联合植物修复是土壤重金属污染修复理论研究和应用实践的新思路。较之根际促生菌,内生菌因生存环境稳定且与植物联系更加紧密,在实际应用中具有更大价值。在重金属胁迫下,部分具有特定功能的细菌可进入植物体内成为内生菌,这些内生菌通常在重金属吸收、耐受和解毒方面具有优良的特性,而且可以协同宿主植物耐受重金属胁迫,表现在直接或间接降低植物体内重金属胁迫强度和提高植物本身对重金属的耐受性两方面。系统分析了内生菌对宿主植物的解毒机制,综述了近年来内生菌增强植物重金属耐受性的研究,展望了重金属胁迫下植物和内生菌互作机制的研究思路和方向。     

Root endophytes enhance stress‐tolerance of Cicuta virosa L. growing in a mining pond of eastern Japan

Cicuta virosa L. plants can grow in a pond subjected to heavy‐metal inputs at the Hitachi mine, eastern Japan. They accumulate heavy‐metal elements, especially high concentrations of zinc (Zn), in their roots. We focused on the role that root bacterial endophytes play in the heavy‐metal uptake of plants and the provision of heavy‐metal tolerance within plants. Our purpose was to clarify the effects of endophytes on: (i) Zn accumulation in C. virosa roots; (ii) growth of C. virosa seedlings; and (iii) heavy‐metal tolerance of C. virosa plants. Root endophytic Pseudomonas putida and Rhodopseudomonas sp., which induced the high production of Zn‐chelating compounds, were selected for the seedling inoculation test. The results of the inoculation test demonstrated that both strains of endophytes increased Zn accumulation in C. virosa roots by solubilizing Zn in the sediment. Both strains also increased the growth of seedlings by possible production of indole‐3‐acetic acid in the plant. The heavy‐metal tolerance of C. virosa seedlings was likely promoted by producing metal‐chelating compounds that detoxify the metals in the plant tissues, and by decreasing the heavy‐metal contents in the tissues via rapid seedling growth. Thus, such mutualistic interactions between plants and bacteria contribute to the persistence of C. virosa in this severe environment.     

Phytoavailability and Leachability of Heavy Metals from Contaminated Soil Treated with Composted Livestock Manure

A study was carried out with maize as the test crop to investigate the bioavailability and leachability of heavy metals (HM) from HM-contaminated soils treated with composted manure. The application of composted manure increased the maize shoot growth by 32.3% and root growth by 30.5% compared with fresh manure. The concentration of HM in maize shoot varied in the order lead (Pb) > nickel (Ni) > zinc (Zn) > chromium (Cr) > copper (Cu) > cadmium (Cd). Whether for the shoot or root, the heavy metal concentrations decreased as the length of manure composting increased, but concentrations were higher in the root than the shoot. Composting decreased the bioavailability and leachability of HM and, hence, their export to the environment more effectively than direct use of fresh manure. Thus, the use of composted manure in place of fresh manure in polluted soils would be more beneficial for mitigating HM pollution.     

Effect of heavy metals combined stress on growth and metals accumulation of three Salix species with different cutting position

This study aimed to compare growth performance and heavy metal (HM) accumulation at different cutting positions of Salix species grown in multi-metal culture. Three Salix species stems cut at different positions (apical to basal) were grown hydroponically for four weeks. The plants were then treated for three weeks with 0, 5, 10, and 20 μM Cd, Cu, Pb, and Zn, resulting in total metal concentrations of 0, 20, 40, and 80 μM. The growth parameters and HM content in shoots and initial cutting were measured. Results showed that, compared with S. fragilis, S. matsudana grew more poorly in uncontaminated condition but grew better and accumulated lower metal in shoots under mixed HM treatment. In addition, cuttings from apical parent stem position exhibited poorer growth performance before and after treatment, as well as greater metal content in shoots than base parts under the HM treatment. These results suggest that S. matsudana may undergo a special mechanism to hinder metals in the initial cutting, thus mitigating growth damage. The apical portion also showed poor resistance against the invasion of mixed HMs because of the immature structure. Therefore, in the selection of phytoremediation plants, metal accumulation ability is not proportional to growth performance.     

Effect of arbuscular mycorrhizal fungal inoculation on heavy metal accumulation of maize grown in a naturally contaminated soil

A pot culture experiment was carried out to study heavy metal (HM) phytoaccumulation from soil contaminated with Cu, Zn, Pb, and Cd by maize (Zea mays L.) inoculated with arbuscular mycorrhizal (AM) fungi (AMF). Two AM fungal inocula--MI containing only one AM fungal strain (Glomus caledonium 90036) and MII consisting of Gigaspora margarita ZJ37, Gigaspora decipens ZJ38, Scutellospora gilmori ZJ39, Acaulospora spp., and Glomus spp.--were applied to the soil under unsterilized conditions. The control received no mycorrhizal inoculation. The maize plants were harvested after 10 wk of growth. MI-treated plants had higher mycorrhizal colonization than MII-treated plants. Both MI and MII increased P concentrations in roots, but not in shoots. Neither MI nor MII had significant effects on shoot or root dry weight (DW). Compared with the control, shoot Cu, Zn, Pb, and Cd concentrations were decreased by MI but increased by MII. Cu, Zn, Pb, and Cd uptake into shoots and roots all increased in MII-treated plants, while in MI-treated plants Cu, Zn, and Pb uptake into shoots and Cd uptake into roots decreased but Cu, Zn, and Pb uptake into roots and Cd into shoots increased. MII was more effective than MI in promoting HM extraction efficiencies. The results indicate that MII can benefit HMphytoextraction and, therefore, show potential in the phytoremediation of HM-contaminated soils.     

植物修复重金属污染及内生细菌效应

土壤和水体的重金属污染已严重危害人类生存环境与健康。由于受重金属污染的环境分布广泛,迫切需要开发经济的清除环境重金属的技术。植物修复是通过绿色植物降解或移除环境污染物,有望成为重金属污染环境的原位修复技术。植物内生菌是指定殖于健康植物的各种组织和器官内部的细菌,被感染的宿主植物不表现出外在病症,耐重金属的内生菌在多种超富集植物中存在。在植物修复过程中,野生型内生菌或基因工程内生菌的抗性系统能降低重金属植物毒性,促进其迁移金属。耐重金属内生菌还可以通过固氮、溶解矿物元素及产生类植物激素、铁载体和ACC脱氨酶等产物促进植物的生长。主要综述目前植物-内生菌相互作用及其潜在的促进植物修复重金属污染的研究进展。     

Endophyte-mediated adjustments in host morphology and physiology and effects on host fitness traits in grasses

Endophytic fungi have been shown to increase tolerance of hosts to biotic and abiotic stresses and in some cases alter growth and development of plants. In this article we evaluate some effects that clavicipitaceous endophytes have on development and physiology of plant tissues. We postulate that oxidative stress protection is the fundamental underlying benefit conferred by many endophytes, accounting for frequently observed enhanced disease resistance, drought tolerance, heavy metal tolerance and tolerance to numerous additional oxidative stresses. We hypothesize that endophyte-mediated oxidative stress protection of the host is the result of at least two processes, including: (1) secretion of reactive oxygen species (ROS) from endophytic mycelia into plant cells; and (2) secretion of auxin from endophytic mycelia into plant cells. Both processes result in an increase in ROS in plant tissues; and stimulate plant tissues to increase activities of antioxidant systems. Auxin is suggested to function in suppression of plant cell death and may be important in maintaining the endophyte–plant symbiosis.     

Endophytic bacteria of the rock-dwelling cactus Mammillaria fraileana affect plant growth and mobilization of elements from rocks

Mammillaria fraileana is a major pioneer, small cactus that harbors endophytic bacteria that have plant growth-promoting traits, including rock-weathering capacity. Our working hypothesis was that this functional group of endophytic bacteria assists in establishing pioneer plants on rocks. When these endophytic bacteria were inoculated on seedlings grown in rock substrate, mobilization of elements from the substrate increased at variable levels across combinations of substrates and inoculants. In plants grown in the rhyodacite substrate, where these cacti naturally grow, increased mobilization occurred in plants inoculated with several strains. Promotion of plant growth, manifested as an increase in dry weight, was greater in cacti inoculated with Enterobacter sakazakii M2PFe. Accumulation of nocturnal acids, indicating photosynthesis by crassulacean acid metabolism, was superior in plants inoculated with the endophytes Azotobacter vinelandii M2Per and Pseudomonas putida M5TSA. Inoculation with endophytes can stimulate plant growth of M. fraileana by mobilizing elements from rock, which can lead to higher photosynthetic activity and accumulation of biomass. Inoculation with P. putida M5TSA also led to accumulation of more total nitrogen than plants inoculated with a control nitrogen-fixing bacteria. Evidence of endophytic colonization is provided after initial inoculation of seedlings and re-isolation and sequencing of 16S DNA of recovered bacteria from developing disinfected plants. The associative interaction between pioneer cacti and their bacterial endophytes enable the host plants to grow in places where plants do not normally grow. Through colonization and establishment of pioneer plants, soil is created, which facilitates colonization by other desert species and contributes to the diversity of dry lands.     

Arbuscular Mycorrhizal Fungi, Bacillus cereus, and Candida parapsilosis from a Multicontaminated Soil Alleviate Metal Toxicity in Plants

We investigated if the limited development of Trifolium repens growing in a heavy metal (HM) multicontaminated soil was increased by selected native microorganisms, bacteria (Bacillus cereus (Bc)), yeast (Candida parapsilosis (Cp)), or arbuscular mycorrhizal fungi (AMF), used either as single or dual inoculants. These microbial inoculants were assayed to ascertain whether the selection of HM-tolerant microorganisms can benefit plant growth and nutrient uptake and depress HM acquisition. The inoculated microorganisms, particularly in dual associations, increased plant biomass by 148% (Bc), 162%, (Cp), and 204% (AMF), concomitantly producing the highest symbiotic (AMF colonisation and nodulation) rates. The lack of AMF colonisation and nodulation in plants growing in this natural, polluted soil was compensated by adapted microbial inoculants. The metal bioaccumulation abilities of the inoculated microorganisms and particularly the microbial effect on decreasing metal concentrations in shoot biomass seem to be involved in such effects. Regarding microbial HM tolerance, the activities of antioxidant enzymes known to play an important role in cell protection by alleviating cellular oxidative damage, such as superoxide dismutase, catalase, glutathione reductase, and ascorbate peroxidase, were here considered as an index of microbial metal tolerance. Enzymatic mechanisms slightly changed in the HM-adapted B. cereus or C. parapsilosis in the presence of metals. Antioxidants seem to be directly involved in the adaptative microbial response and survival in HM-polluted sites. Microbial inoculations showed a bioremediation potential and helped plants to develop in the multicontaminated soil. Thus, they could be used as a biotechnological tool to improve plant development in HM-contaminated environments.     

南通沿海滩涂耐盐植物重金属抗性内生细菌的筛选及生物多样性

【目的】沿海滩涂耐盐植物重金属抗性内生细菌的筛选及其促生长潜在能力的研究有助于我们获得一些能够耐受并促进耐盐植物在被Cd2+、Pb2+、Hg2+、Cu2+,Zn2+等重金属离子污染的贫瘠的沿海滩涂上正常生长的菌株,达到既能够利用广袤的滩涂生物资源产生经济价值又能够净化生态环境的目的。【方法】以江苏南通沿海滩涂地区的4种耐盐植物为材料,采用稀释平板涂布法从中分离得到45株内生细菌,从中挑取23株代表性的菌株,对其进行抗重金属Cu2+、Pb2+、Cd2+、Zn2+,Hg2+的活性筛选;固氮、解磷、吲哚乙酸(IAA)的产生、1-氨基环丙烷-1-羧酸(ACC)脱氨酶活性等促生指标以及NaCl耐受能力的筛选。【结果】发现分离所得的大多数细菌能够耐受高浓度的Cu2+以及Pb2+,但是对Cd2+、Zn2+,Hg2+的耐受能力则较弱;26.1%的细菌具有固氮能力,21.7%的细菌具有解磷能力,60.9%的细菌能够产生IAA,39.1%的细菌含有ACC脱氨酶。对他们进行16S rRNA基因鉴定后发现,他们分属于芽胞杆菌属(Bacillus)、喜盐芽胞杆菌属(Halobacillus)、海洋芽胞杆菌属(Oceanobacillus)、微小杆菌属(Exiguobacterium)、沙雷氏菌属(Serratia)、短波单胞菌属(Brevundimonas)、弧菌属(Vibrio)、葡萄球菌属(Staphylococcus)共8个属,显示了丰富的多样性。其中菌株KLBMP 2432以及菌株KLBMP 2447为潜在的新种。【结论】沿海滩涂地区的耐盐植物内生细菌具有丰富多样的生物多样性以及促生长能力,且存在潜在的新种资源,并对重金属Cu2+,Pb2+具有较强的抗性。     

Burkholderia sp. SCMS54 reduces cadmium toxicity and promotes growth in tomato

Cadmium (Cd) can enter soil through the use of fertilisers, calcareous, pesticides and industrial and/or domestic effluents. Cd can leach into groundwater and be taken up by plants, potentially leading to reductions in plant growth and yield. In soil, plant roots interact with heavy metal (HM)‐tolerant microorganisms that may promote plant growth. Soil microorganisms may also be able to solubilise or mobilise soil metals, thereby acting as bioremediators. A better understanding of the interaction among plants, metals, microorganisms and soil will lead to improved plant tolerance. Two multi‐tolerant bacteria from the Burkholderia genus were isolated from Cd‐contaminated and Cd‐uncontaminated soil of a coffee plantation. In addition to its high tolerance to Cd, the strain SCMS54 produces indole‐acetic acid (IAA), solubilises inorganic phosphate and produces siderophores, demonstrating its potential to contribute to beneficial plant–microorganism interactions. When interacting with tomato plants exposed to Cd, the bacterium led to decreases in plant peroxide and chlorosis levels, promoted relative plant growth and decreased the root absorption of Cd, resulting in increased plant tolerance to this highly toxic HM. The results indicated that the inoculation of tomato plants with Burkholderia sp. SCMS54 promotes better growth in plants cultivated in the presence of Cd. This phenomenon appears to be attributed to a mechanism that decreases Cd concentrations in the roots via a beneficial interaction between the bacteria and the plant roots.     

玉米内生细菌资源研究进展

植物内生菌存在于植物的各个器官,种类繁多,对植物的生长发育具有重要作用。玉米是最重要粮食作物之一,通过研究玉米与其内生细菌的关系,对增产玉米以及生物防治有积极意义。本文归纳了玉米内生细菌资源的分离与收集,包括内生细菌的多样性及新种的鉴定;介绍了玉米内生细菌与宿主的相关性;总结了玉米内生细菌的生物学作用,包括抑菌性、耐受性、固氮作用和促生作用,以及内生细菌对玉米作用机制;并对玉米内生细菌研究趋势进行了前景展望,旨在为玉米内生细菌的研究提供参考。     

植物螯合肽及其在重金属耐性中的作用

综述植物螯合肽的生物合成及其在重金属耐性中的作用．有毒重金属在土壤中的积累不仅影响作物的生长和产量形成,而且严重威胁农产品的安全性．植物对重金属的耐性和积累在种间和基因型之间存在着很大的差异,在重金属胁迫条件下植物螯合肽(PC)的合成是植物对胁迫的一种适应性反应,耐性基因型合成较多的PC谷胱苷肽是合成PC的前体,PC可与重金属螯合,并进一步转运至液泡贮存,使细胞质的重金属浓度降低,从而达到解毒效果．重金属诱导植物合成PC的遗传机理和生化途径有赖于分子生物学的深入研究,cD-敏感型拟南芥突变体Cad1-1(缺失GSH)和Cad2-1(缺失PC合成诱导酶)的分离及相关研究,佐证了PC在Cd-解毒中起关键作用．对PC在重金属污染土壤或水体的植物修复和农作物安全生产中的意义进行了讨论．     

种子内生菌增强宿主植物重金属抗性的功能机制研究进展

种子是植物的繁殖器官,其内定殖有一定数量的内生菌,种子内生菌通过垂直传播成为新生植物组织内最早定殖的微生物,对连续几代植物内生菌群落的形成起着决定性作用,并在植物抗逆方面发挥着重要作用。本文对种子内生菌与宿主植物重金属抗性之间的关系及其功能机制进行综述,并对下一步研究方向予以展望。     

Physiological and molecular mechanisms of heavy metal accumulation in nonmycorrhizal versus mycorrhizal plants

Uptake, translocation, detoxification, and sequestration of heavy metals (HMs) are key processes in plants to deal with excess amounts of HM. Under natural conditions, plant roots often establish ecto‐ and/or arbuscular‐mycorrhizae with their fungal partners, thereby altering HM accumulation in host plants. This review considers the progress in understanding the physiological and molecular mechanisms involved in HM accumulation in nonmycorrhizal versus mycorrhizal plants. In nonmycorrhizal plants, HM ions in the cells can be detoxified with the aid of several chelators. Furthermore, HMs can be sequestered in cell walls, vacuoles, and the Golgi apparatus of plants. The uptake and translocation of HMs are mediated by members of ZIPs, NRAMPs, and HMAs, and HM detoxification and sequestration are mainly modulated by members of ABCs and MTPs in nonmycorrhizal plants. Mycorrhizal‐induced changes in HM accumulation in plants are mainly due to HM sequestration by fungal partners and improvements in the nutritional and antioxidative status of host plants. Furthermore, mycorrhizal fungi can trigger the differential expression of genes involved in HM accumulation in both partners. Understanding the molecular mechanisms that underlie HM accumulation in mycorrhizal plants is crucial for the utilization of fungi and their host plants to remediate HM‐contaminated soils.     

Phytoremediation of Heavy Metals: Physiological and Molecular Mechanisms

Heavy metals (HM) are a unique class of toxicants since they cannot be broken down to non-toxic forms. Concentration of these heavy metals has increased drastically, posing problems to health and environment, since the onset of the industrial revolution. Once the heavy metals contaminate the ecosystem, they remain a potential threat for many years. Some technologies have long been in use to remove, destroy and sequester these hazardous elements. Even though effective techniques for cleaning the contaminated soils and waters are usually expensive, labour intensive, and often disturbing. Phytoremediation, a fast-emerging new technology for removal of toxic heavy metals, is cost-effective, non-intrusive and aesthetically pleasing. It exploits the ability of selected plants to remediate pollutants from contaminated sites. Plants have inter-linked physiological and molecular mechanisms of tolerance to heavy metals. High tolerance to HM toxicity is based on a reduced metal uptake or increased internal sequestration, which is manifested by interaction between a genotype and its environment. The growing interest in molecular genetics has increased our understanding of mechanisms of HM tolerance in plants and many transgenic plants have displayed increased HM tolerance. Improvement of plants by genetic engineering, i.e., by modifying characteristics like metal uptake, transport and accumulation and plant’s tolerance to metals, opens up new possibilities of phytoremediation. This paper presents an overview of the molecular and physiological mechanisms involved in the phytoremediation process, and discusses strategies for engineering plants genetically for this purpose.     

Enhancement of the efficiency of Cd phytoextraction using bacterial endophytes isolated from Chromolaena odorata,a Cd hyperaccumulator

Phytoextraction is a technique using a hyperaccumulator to remove heavy metals from soil. The efficiency of heavy metal uptake can be enhanced by the inoculation of endophytes. In this study, we isolated and identified 23 endophytes from Chromolaena odorata, a cadmium (Cd) hyperaccumulator that consisted of 19 bacteria, 2 actinomycetes and 2 fungi. All bacteria and fungi could produce at least 1 plant growth promoting factors. However, only 4 bacterial isolates; Paenibacillus sp. SB12, Bacillus sp. SB31, Bacillus sp. LB51, and Alcaligenes sp. RB54 showed the highest minimum inhibitory concentration (MIC) value (2.9 mM), followed by Exiguobacterium sp.RB51 (1.7 mM). Then, these 5 high-MIC bacteria and 1 low-MIC bacterium, Bacillus sp. LB15 were inoculated onto sunflower grown in soil supplemented with 250 mg/kg of Cd. After 60 days, all inoculated plants accumulated significantly higher Cd concentration than the non-inoculated counterparts, and those inoculated with strain LB51 showed the highest Cd accumulation and growth. Interestingly, strain LB15 with low MIC also enhanced Cd accumulation in plants. The results suggest that these bacteria, particularly strain LB51, could be applied to improve Cd accumulation in plants, and that bacteria with low MIC also have the potential to enhance the efficiency of phytoextraction.     

More than 400 million years of evolution and some plants still can't make it on their own: plant stress tolerance via fungal symbiosis

All plants in natural ecosystems are thought to be symbioticwith mycorrhizal and/or endophytic fungi. Collectively, thesefungi express different symbiotic lifestyles ranging from parasitismto mutualism. Analysis of Colletotrichum species indicates thatindividual isolates can express either parasitic or mutualisticlifestyles depending on the host genotype colonized. The endophytecolonization pattern and lifestyle expression indicate thatplants can be discerned as either disease, non-disease, or non-hosts.Fitness benefits conferred by fungi expressing mutualistic lifestylesinclude biotic and abiotic stress tolerance, growth enhancement,and increased reproductive success. Analysis of plant–endophyteassociations in high stress habitats revealed that at leastsome fungal endophytes confer habitat-specific stress toleranceto host plants. Without the habitat-adapted fungal endophytes,the plants are unable to survive in their native habitats. Moreover,the endophytes have a broad host range encompassing both monocotsand eudicots, and confer habitat-specific stress tolerance toboth plant groups. Key words: Colletotrichum, fungal endophytes, stress tolerance, symbiosis, symbiotic lifestyle Received 19 June 2007; Revised 25 November 2007 Accepted 30 November 2007