Induction of abiotic stress tolerance in plants by endophytic microbes

Endophytes are micro‐organisms including bacteria and fungi that survive within healthy plant tissues and promote plant growth under stress. This review focuses on the potential of endophytic microbes that induce abiotic stress tolerance in plants. How endophytes promote plant growth under stressful conditions, like drought and heat, high salinity and poor nutrient availability will be discussed. The molecular mechanisms for increasing stress tolerance in plants by endophytes include induction of plant stress genes as well as biomolecules like reactive oxygen species scavengers. This review may help in the development of biotechnological applications of endophytic microbes in plant growth promotion and crop improvement under abiotic stress conditions.

Significance and Impact of the Study

Increasing human populations demand more crop yield for food security while crop production is adversely affected by abiotic stresses like drought, salinity and high temperature. Development of stress tolerance in plants is a strategy to cope with the negative effects of adverse environmental conditions. Endophytes are well recognized for plant growth promotion and production of natural compounds. The property of endophytes to induce stress tolerance in plants can be applied to increase crop yields. With this review, we intend to promote application of endophytes in biotechnology and genetic engineering for the development of stress‐tolerant plants.     

The plant endosphere world – bacterial life within plants

The plant endosphere is colonized by complex microbial communities and microorganisms, which colonize the plant interior at least part of their lifetime and are termed endophytes. Their functions range from mutualism to pathogenicity. All plant organs and tissues are generally colonized by bacterial endophytes and their diversity and composition depend on the plant, the plant organ and its physiological conditions, the plant growth stage as well as on the environment. Plant-associated microorganisms, and in particular endophytes, have lately received high attention, because of the increasing awareness of the importance of host-associated microbiota for the functioning and performance of their host. Some endophyte functions are known from mostly lab assays, genome prediction and few metagenome analyses; however, we have limited understanding on in planta activities, particularly considering the diversity of micro-environments and the dynamics of conditions. In our review, we present recent findings on endosphere environments, their physiological conditions and endophyte colonization. Furthermore, we discuss microbial functions, the interaction between endophytes and plants as well as methodological limitations of endophyte research. We also provide an outlook on needs of future research to improve our understanding on the role of microbiota colonizing the endosphere on plant traits and ecosystem functioning.     

Bacterial endophytes from arid land plants regulate endogenous hormone content and promote growth in crop plants: an example of Sphingomonas sp. and Serratia marcescens

The objective of the present study was to determine the potential plant growth-promoting action of bacterial endophytes isolated from arid land-dwelling plants under normal conditions. Overall, five bacterial endophytes LK11 (Sphingomonas sp. LK11), TP5 (Bacillus subtilis), MPB5.3 (B. subtilis subsp. Subtilis), S9 (B. subtilis subsp. Subtilis), and TP1 (Serratia marcescens) were evaluated based on morphological characteristics after isolation and purification. Phytohormonal analysis of these endophytes predicted indole acetic acid (IAA) production 12.31?±?0.45?, 6.8?±?0.59, and 10.5?±?1.02?μM/mL in the culture broths of LK11, MPB5.3, and TP1, respectively. Under controlled greenhouse conditions, these endophytes were inoculated into soybean, and their growth-promoting characteristics were compared with those of non-phytohormone-producing endophytes. In terms of plant growth promotion, among IAA-producing endophytes, LK11 and TP1 greatly improved physiological characteristics such as shoot/root length, fresh/dry weight, and chlorophyll contents. However, the non-phytohormone-producing endophytes TP5 and S9 did not show a growth-promoting effect. Based on these results, plants inoculated with LK11 and TP1 along with a control were subjected to endogenous hormonal analysis and showed a significant increase in abscisic acid (457.30–398.55 vs. 205.93 ng/g D.W.) and a decrease in jasmonic acid content (50.07–85.07 vs. 93.90 ng/g D.W.), respectively. Total gibberellin content was found to significantly increase in endophyte-inoculated plants (155.43–146.94?ng/g D.W.) as compared to that in controls (113.76 ng/g D.W.). In summary, bacterial endophytes might be used to enhance crop plant physiological characteristics isolated from arid land-inhabiting plants under normal conditions.     

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.     

Increased fitness of rice plants to abiotic stress via habitat adapted symbiosis: a strategy for mitigating impacts of climate change

Climate change and catastrophic events have contributed to rice shortages in several regions due to decreased water availability and soil salinization. Although not adapted to salt or drought stress, two commercial rice varieties achieved tolerance to these stresses by colonizing them with Class 2 fungal endophytes isolated from plants growing across moisture and salinity gradients.Plant growth and development, water usage, ROS sensitivity and osmolytes were measured with and without stress under controlled conditions.The endophytes conferred salt, drought and cold tolerance to growth chamber and greenhouse grown plants. Endophytes reduced water consumption by 20-30% and increased growth rate, reproductive yield, and biomass of greenhouse grown plants. In the absence of stress, there was no apparent cost of the endophytes to plants, however, endophyte colonization decreased from 100% at planting to 65% compared to greenhouse plants grown under continual stress (maintained 100% colonization).These findings indicate that rice plants can exhibit enhanced stress tolerance via symbiosis with Class 2 endophytes, and suggest that symbiotic technology may be useful in mitigating impacts of climate change on other crops and expanding agricultural production onto marginal lands.     

Characterization of bacterial endophytes of sweet potato plants

Endophytic bacteria associated with sweet potato plants (Ipomoea batatas (L.) Lam.) were isolated, identified and tested for their ability to fix nitrogen, produce indole acetic acid (IAA), and exhibit stress tolerance. Eleven different strains belonging to the genera, Enterobacter, Rahnella, Rhodanobacter, Pseudomonas, Stenotrophomonas, Xanthomonas and Phyllobacterium, were identified. Four strains were shown to produce IAA (a plant growth hormone) and one strain showed the ability to grow in nitrogen free medium and had the nitrogenase subunit gene, nifH. To determine if IAA production by the endophytes had any role in protecting the cells against adverse conditions, different stress tests were conducted. The IAA producer grew well in the presence of some antibiotics, UV and cold treatments but the response to pH, osmotic shock, thermal and oxidative treatments was the same for both the IAA producer and the no IAA producer. To determine if IAA produced by the strains was biologically relevant to plants, cuttings of poplar were inoculated with the highest IAA producing strain. The inoculated cuttings produced roots sooner and grew more rapidly than uninoculated cuttings. These studies indicate that endophytes of sweet potato plants are beneficial to plant growth.     

Intraspecific competition of endophyte infected vs uninfected plants of two woodland grass species

Grass endophytes (Clavicipitaceae, Ascomycota) are generally considered to be mutualists which increase the host's fitness. Infected plants are often more persistent and competitive than uninfected plants, influencing population dynamics and plant community diversity. However, most of this empirical evidence is based on studies focusing on agronomically important grass species such as tall fescue or perennial ryegrass and their implications for livestock and man-made habitats. Recent studies indicate that endophyte-plant associations may be more variable, ranging from parasitic to mutualistic. In the present study, we investigated the influence of endophyte infection on two wild woodland grasses, which are naturally infected with distinct fungal endophytes: Brachypodium sylvaticum with Epichloë sylvatica and Bromus benekenii with Epichloë bromicola . An intraspecific competition experiment was conducted over two growing seasons in the greenhouse and in an experimental garden. At first harvest (after 12 weeks growing), endophyte infection had a significant negative effect on above ground dry matter yield (DMY) of B. sylvaticum , but a significant positive effect on DMY of Br. benekenii under competition. The same differential effects on DMY and on total seed number were also observed at final harvest (after 62 weeks growing). Results from Br. benekenii were consistent with our hypothesis of increased competitive abilities of infected plants in nature which could explain the high infection rate observed in natural populations. In contrast, this explanation does not hold true for B. sylvaticum , and other factors such as increased herbivore and pathogen resistance together with frequent horizontal transmission may be responsible for the very high incidence of this association in nature. Our results confirm previous predictions that beneficial effects of endophyte infection in wild grasses can vary for different grass species, even in comparable habitats.     

Drought-induced responses of photosynthesis and antioxidant metabolism in higher plants

Environmental stresses trigger a wide variety of plant responses, ranging from altered gene expression and cellular metabolism to changes in growth rates and crop yields. A plethora of plant reactions exist to circumvent the potentially harmful effects caused by a wide range of both abiotic and biotic stresses, including light, drought, salinity, high temperatures, and pathogen infections. Among the environmental stresses, drought stress is one of the most adverse factors of plant growth and productivity. Understanding the biochemical and molecular responses to drought is essential for a holistic perception of plant resistance mechanisms to water-limited conditions. Drought stress progressively decreases CO2 assimilation rates due to reduced stomatal conductance. Drought stress also induces reduction in the contents and activities of photosynthetic carbon reduction cycle enzymes, including the key enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase. The critical roles of proline and glycine-betaine, as well as the role of abscisic acid (ABA), under drought stress conditions have been actively researched to understand the tolerance of plants to dehydration. In addition, drought stress-induced generation of active oxygen species is well recognized at the cellular level and is tightly controlled at both the production and consumption levels in vivo, through increased antioxidative systems. Knowledge of sensing and signaling pathways, including ABA-mediated changes in response to drought stress, is essential to improve crop management. This review focuses on the ability and strategies of higher plants to respond and adapt to drought stress.     

Arbuscular mycorrhizal symbiosis alleviates detrimental effects of saline reclaimed water in lettuce plants

The present study evaluated the effects of inoculation with arbuscular mycorrhizal fungi (AMF; Glomus iranicum var. tenuihypharum sp. nova) on the physiological performance and production of lettuce plants grown under greenhouse conditions and supplied with reclaimed water (RW; urban-treated wastewater with high electrical conductivity; 4.19 dS m^?1). Four treatments, fresh water, fresh water plus AMF inoculation, RW and RW plus AMF inoculation, were applied and their effects, over time, analyzed. Root mycorrhizal colonization, plant biomass, leaf-ion content, stomatal conductance and net photosynthesis were assessed. Overall, our results highlight the significance of the AMF in alleviation of salt stress and their beneficial effects on plant growth and productivity. Inoculated plants increased the ability to acquire N, Ca, and K from both non-saline and saline media. Moreover, mycorrhization significantly reduced Na plant uptake. Under RW conditions, inoculated plants also showed a better performance of physiological parameters such as net photosynthesis, stomatal conductance and water-use efficiency than non-mycorrhizal plants. Additionally, the high concentration of nutrients already dissolved in reclaimed water suggested that adjustments in the calculation of the fertigation should be conducted by farmers. Finally, this experiment has proved that mycorrhization could be a suitable way to induce salt stress resistance in iceberg lettuce crops as plants supplied with reclaimed water satisfied minimum legal commercial size thresholds. Moreover, the maximum values of Escherichia coli in the reclaimed water were close to but never exceeded the international thresholds established (Spanish Royal Decree 1620/2007; Italian Decree, 2003) and hence lettuces were apt for sale.     

Prospectives for applying molecular and genetic methodology to improve wheat cultivars in drought environments

With the advent of molecular biotechnologies, new opportunities are available for plant physiologists to study the relationships between wheat traits and their genetic control. The functional determinations of all genes that participate in drought adaptation or tolerance reactions are expected to provide an integrated understanding of the biochemical and physiological basis of stress responses in wheat. However, despite all the recent technological breakthroughs, the overall contribution of genomics-assisted breeding to the release of drought-resilient wheat cultivars has so far been marginal. This paper critically analyses how biotechnological, genetic and information tools can contribute to accelerating the release of improved, drought-tolerant wheat cultivars. Armed with such information from established models, it will be possible to elucidate the physiological basis of drought tolerance and to select genotypes with an improved yield under water-limited conditions.     

Effects of cross host species inoculation of nitrogen‐fixing endophytes on growth and leaf physiology of maize

Plants that grow and thrive under abiotic stress often do so with the help of endophytic microorganisms. Although nitrogen‐fixing (diazotrophic) endophytes colonize many wild plants, these natural relationships may be disrupted in cultivated crop species where breeding and genotype selection often occur under conditions of intensive fertilization and irrigation. Many energy crops including corn may still benefit from diazotrophic endophyte inoculations allowing for more efficient biomass production with less input of petroleum‐derived fertilizer. A selection of diazotrophic endophytes isolated from willow (Salix sitchensis, Sitka willow) and poplar (Populus trichocarpa, black cottonwood) growing in nutrient‐poor river sides were used as inoculum in three experiments testing the effect on plant growth and leaf level physiology of a sweet corn variety under various levels of applied nitrogen fertilizer. We report substantial growth promotion with improved leaf physiology of corn plants in response to diazotrophic endophyte inoculations. Significant gains of early biomass with a greater root : shoot ratio were found for plants receiving endophytic inocula over the uninoculated control groups regardless of the nitrogen level. Furthermore, inoculated plants exhibited consistently higher rates of net CO₂ assimilation than did those without endophytic inoculation. These results have beneficial implications for enhanced plant growth in a low‐input system on nutrient‐poor sites. The immediate increase of root mass observed in endophyte inoculated plants has the potential to provide better establishment and early growth in resource‐limited environments. The initial results of this study also indicate that the beneficial effect from endophytes isolated from poplar and willow species is not restricted to the species from which they were initially isolated.     

A meta-analysis on morphological,physiological and biochemical responses of plants with PGPR inoculation under drought stress

Plant growth-promoting rhizobacteria (PGPR) can help plants to resist drought stress. However, the mechanisms of how PGPR inoculation affect plant status under drought remain incompletely understood. We performed a meta-analysis of plant response to PGPR inoculation by compiling data from 57 PGPR-inoculation studies, including 2, 387 paired observations on morphological, physiological and biochemical parameters under drought and well-watered conditions. We compare the PGPR effect on plants performances among different groups of controls and treatments. Our results reveal that PGPR enables plants to restore themselves from drought-stressed to near a well-watered state, and that C4 plants recover better from drought stress than C3 plants. Furthermore, PGPR is more effective underdrought than well-watered conditions in increasing plant biomass, enhancing photosynthesis and inhibiting oxidant damage, and the responses of C4 plants to the PGPR effect was stronger than that of C3 plants under drought conditions. Additionally, PGPR belonging to different taxa and PGPR with different functional traits have varying degrees of drought-resistance effects on plants. These results are important to improve our understanding of the PGPR beneficial effects on enhanced drought-resistance of plants.     

The impact of the endophytic bacterial community on mulberry tree growth in the Three Gorges Reservoir ecosystem,China

Plant-associated microbes influence plant performance and may also impact biotic and abiotic stress tolerance. The microbiome of mulberry trees planted for ecological restoration in the hydro-fluctuation belt of the Three Gorges Reservoir Region, China, exhibited distinct patterns of localization. The endosphere exhibited lower α-diversity relative to the rhizosphere, but was more closely related to host growth status, especially in stem tissues. Pantoea was the predominant bacterial genus inhabiting the stems of two well-growing plants, while sequences identified as Pseudomonas and Pantoea were abundant in poorly growing plants. The complexity of the endophytic community was more connected to growth status in well-growing plants than it was in poorly growing plants. Among 151 endophytes cultured from collected samples of mulberry, 64 exhibited plant growth-promoting (PGP) potential in vitro and the majority of beneficial taxa were harvested from well-growing plants. Collectively, the present study indicates that the recruitment of beneficial endophytes may contribute to mulberry fitness under abiotic stress, and it provides a foundation for the development of a new strategy in vegetation restoration.     

Overexpression of DnaK from a halotolerant cyanobacterium Aphanothece halophytica enhances growth rate as well as abiotic stress tolerance of poplar plants

The DnaK/Hsp70 family is a molecular chaperone that binds non-native states of other proteins, and concerns to various physiological processes in the bacterial, plant and animal cells. Previously, we showed that overexpression of DnaK from a halotolerant cyanobacterium Aphanothece halophytica (ApDnaK) enhances tolerance to abiotic stresses such as high salinity and high temperature in tobacco plants. Here, we tested the transformation of poplar (Populus alba) with ApDnaK for enhancing the growth of transformed poplar plants. Under control growth conditions, transgenic poplar plants exhibited similar growth rates with the wild-type plants during young seedlings under low light intensity, whereas they showed faster growth, larger plant size, and higher cellulose contents when poplar plants were grown under high light intensity. Transgenic young poplar plants exhibited more rapid recovery from the stresses of high salinity, drought, and low temperature compared with those of the wild type plants when poplar plants were grown under low light intensity. These results suggest that ApDnaK could be useful to enhance the growth rate as well as to increase the stress tolerance.     

Overexpression of a wheat Na+/H+ antiporter gene (TaNHX2) enhances tolerance to salt stress in transgenic tomato plants (Solanum lycopersicum L.)

Soil salinity is a major environmental stress limiting plant productivity. Vacuole Na⁺/H⁺ antiporters play important roles for the survival of plants under salt stress conditions. We have developed salt stress tolerant transgenic tomato plants (Solanum lycopersicum cv. PED) by overexpression of the wheat Na⁺/H⁺ antiporter gene TaNHX2 using Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBin438 that contains the TaNHX2 gene under the control of double CaMV 35S promoter and npt II as a selectable marker. PCR and Southern blot analysis confirmed that TaNHX2 gene has been integrated and expressed in the T₁ generation transgenic tomato plants. When TaNHX2 expressing plants were exposed to 100 or 150 mM NaCl, they were found to be more tolerant to salt stress compared to wild type plants. Biochemical analyses also showed that transgenic plants have substantial amount of relative water content and chlorophyll content under salt stress conditions compared to wild type plants. The relative water content in transgenic and wild type plants ranged from 68 to 75 % and 46–73 % and chlorophyll content fall in between 1.8 to 2.4 mg/g fw and 1.0 to 2.4 mg/g fw, respectively, in all stress conditions. In the present study, we observed a better germination rate of T₁ transgenic seeds under salt stress conditions compared with wild type plants. Our results indicated that TaNHX2-transgenic tomato plants coped better with salt stress than wild type plants.     

The interactive effects of plant microbial symbionts: a review and meta-analysis

In nature, plants often associate with multiple symbionts concurrently, yet the effects of tripartite symbioses are not well understood. We expected synergistic growth responses from plants associating with functionally distinct symbionts. In contrast, symbionts providing similar benefits to a host may reduce host plant growth. We reviewed studies investigating the effect of multiple interactions on host plant performance. Additionally, we conducted a meta-analysis on the studies that performed controlled manipulations of the presence of two microbial symbionts. Using response ratios, we investigated the effects on plants of pairs of symbionts (mycorrhizal fungi, fungal endophytes, and nitrogen-fixers). The results did not support the view that arbuscular mycorrhizal (AM) fungi and rhizobia should interact synergistically. In contrast, we found the joint effects of fungal endophytes and arbuscular mycorrhizal fungi to be greater than expected given their independent effects. This increase in plant performance only held for antagonistic endophytes, whose negative effects were alleviated when in association with AM fungi, while the impact of beneficial endophytes was not altered by infection with AM fungi. Generalizations from the meta-analysis were limited by the substantial variation within types of interactions and the data available, highlighting the need for more research on a range of plant systems.     

Cd、Pb复合处理下2种离子在植物体内的分布及其对植物生理指标的影响

以黄反、小麦为试验材料,以Cd、Pb复合浓度进行处理,研究了在不同处理水平下Cd、Pb在2种植物体内的分布,以及2种植物的叶绿素、脯氨酸含量、乙烯释放量的变化。结果表明,Cd、Pb主要累积于植物根部,不同复合浓度处理后,植物叶片中叶绿素含量下降,脯氨酸含量增高,乙烯释放量增加,并存在峰点。不同的植物及不同的浓度组合有着不同的影响,环境中Cd、Pb对植物的影响存在着相互作用。     

Alleviation of Field Low-Temperature Stress in Winter Wheat by Exogenous Application of Salicylic Acid

Low temperature in later spring severely limits plant growth and causes considerable yield loss in wheat. In this study, the impacts of exogenous salicylic acid (SA) on plant growth, grain yield and key physiological parameters of wheat plants were investigated under field low-temperature conditions using a field air temperature control system (FATC). The results showed that low-temperature stress significantly decreased leaf net photosynthetic rate, plant height and biomass production of wheat plants at the jointing stage, resulting in a reduction in grain yield. Moreover, the growth period of wheat plants was prolonged by low-temperature stress. However, SA-treated plants significantly improved the photochemical efficiency of photosystem II, accumulation of osmo-protectants, activities of enzymatic antioxidants, and pool of non-enzymatic low molecular substances compared with non-SA-treated plants under low-temperature stress. Pretreatment with SA effectively alleviated low-temperature-induced reduction in leaf net photosynthetic rate, plant height, biomass production and grain yield as well as prolonging of growth period of wheat plants. However, SA-treated plants had no significant effects on the expression levels of cold-responsive genes compared with non-SA-treated plants under low-temperature stress. Our results demonstrated that exogenous application of SA is an appropriate strategy for wheat to resist late spring low-temperature stress under field conditions.

     

Anaerobic nitrogen-fixing consortia consisting of clostridia isolated from gramineous plants

We report here the existence of anaerobic nitrogen-fixing consortia (ANFICOs) consisting of N(2)-fixing clostridia and diverse nondiazotrophic bacteria in nonleguminous plants; we found these ANFICOs while attempting to overcome a problem with culturing nitrogen-fixing microbes from various gramineous plants. A major feature of ANFICOs is that N(2) fixation by the anaerobic clostridia is supported by the elimination of oxygen by the accompanying bacteria in the culture. In a few ANFICOs, nondiazotrophic bacteria specifically induced nitrogen fixation of the clostridia in culture. ANFICOs are widespread in wild rice species and pioneer plants, which are able to grow in unfavorable locations. These results indicate that clostridia are naturally occurring endophytes in gramineous plants and that clostridial N(2) fixation arises in association with nondiazotrophic endophytes.