Uninoculated Seedlings Research Articles

Enhancing plant quality and outplanting growth ofAcacia auriculiformisin dry wasteland plantations by inoculating a selected microbial consortium in the nursery

In this study, the performance of a selected microbial consortium (Scutellospora calospora + Azotobacter chroococcum + Bacillus coagulans + Trichoderma harzianum) on Acacia auriculiformis A. Cunn. ex Benth. was evaluated through large-scale nursery trials at three locations in the Mandya district of Karnataka state, India. At each location, 500 inoculated and 500 uninoculated seedlings were cultivated. The increase in plant dry biomass of inoculated plants was 31% (mean of three locations) compared with uninoculated plants. The seedlings inoculated with microbial consortium under large-scale nursery trials were planted in wasteland at three locations, and their growth was monitored for nearly 6 years. At the end of the study, field growth of inoculated trees, measured as the biovolume index, was 52% higher than that of uninoculated trees. This study shows that the selected microbial consortium enhances nursery quality and midterm field growth of Acacia auriculiformis plantations on dry wasteland.

Colonization of Trichoderma viride Tv-1511 in peppermint (Mentha × piperita L.) roots promotes essential oil production by triggering ROS-mediated MAPK activation

Peppermint (Mentha × piperita L.) is a flavoring additive used worldwide, and Trichoderma species are beneficial fungi that can stimulate growth and disease resistance of these plants. Here the growth conditions and metabolic processes of essential oil (EO) biosynthesis in response to inoculation with Trichoderma viride Tv-1511 were investigated. The results showed that T. viride Tv-1511 was able to colonize roots of peppermint to promote its growth and photosynthetic activity and induce higher levels of glandular trichomes and elevated EO yield and composition. GC-MS analysis showed that T. viride Tv-1511-inoculated peppermint produced higher concentrations of menthone, menthol, and pulegone and lower concentrations of menthofuran than un-inoculated seedlings, and qRT-PCR showed that T. viride Tv-1511 inoculation induced upregulation of Pr (pulegone reductase encoding gene) and Mr (menthone reductase encoding gene), whereas it led to the downregulation of Mfs (menthofuran synthase encoding gene). Furthermore, a mitogen-activated protein kinase (MAPK) in peppermint, which was determined to be an analog of Arabidopsis MPK6 protein, was found to be responsible for the modulation of EO metabolism at the transcriptional level and for enzymatic activation in the T. viride Tv-1511-inoculated peppermint. Notably, NADPH oxidase-dependent reactive oxygen species (ROS) production played vital roles in the root colonization of T. viride Tv-1511 and was also involved in the induction of MAPK activation. These data showed the beneficial effects of T. viride Tv-1511 on the seedling growth and EO yield of peppermint, and they elucidated that T. viride Tv-1511 improved the quantity and quality of EOs by regulating the genes that encode the enzymes involved in EO metabolism through a potential MAPK-mediated signaling pathways.

Plant Growth Promotion Abilities of Phylogenetically Diverse Mesorhizobium Strains: Effect in the Root Colonization and Development of Tomato Seedlings.

Mesorhizobium contains species widely known as nitrogen-fixing bacteria with legumes, but their ability to promote the growth of non-legumes has been poorly studied. Here, we analyzed the production of indole acetic acid (IAA), siderophores and the solubilization of phosphate and potassium in a collection of 24 strains belonging to different Mesorhizobium species. All these strains produce IAA, 46% solubilized potassium, 33% solubilize phosphate and 17% produce siderophores. The highest production of IAA was found in the strains Mesorhizobium ciceri CCANP14 and Mesorhizobium tamadayense CCANP122, which were also able to solubilize potassium. Moreover, the strain CCANP14 showed the maximum phosphate solubilization index, and the strain CCANP122 was able to produce siderophores. These two strains were able to produce cellulases and cellulose and to originate biofilms in abiotic surfaces and tomato root surface. Tomato seedlings responded positively to the inoculation with these two strains, showing significantly higher plant growth traits than uninoculated seedlings. This is the first report about the potential of different Mesorhizobium species to promote the growth of a vegetable. Considering their use as safe for humans, animals and plants, they are an environmentally friendly alternative to chemical fertilizers for non-legume crops in the framework of sustainable agriculture.

Ectomycorrhizae determine chestnut seedling growth and drought response

Although agriculture focuses on row crops in the Midwestern US, chestnut (Castanea) would be a possible multipurpose agroforestry crop in erodible locations within this region. As ectomycorrhizal (ECM) colonization is often crucial for tree establishment and production, we addressed the importance of ECM colonization on chestnut performance by assessing: (1) natural ECM colonization in an established chestnut orchard, and (2) the effect of ECM inoculation on seedling establishment and drought response in a greenhouse. We selected 50 Chinese American hybrid chestnuts trees in a commercial orchard and assessed their level of mycorrhizal colonization in relation to distance from an adjacent forest, the source of inoculum. In the green house, we grew 80 seedlings from this orchard with and without mycorrhizal inoculation. Half were harvested to assess inoculation success and biomass; the remainder were subjected to experimental water stress which was evaluated with chlorophyll fluorescence using Fv/Fm values. In the orchard planted with uninoculated plants, ECM colonization decreased with distance from adjacent oak forest (P = 0.015), the putative ECM source, with an average of 29% root tips colonized. ECM inoculation increased seedling aboveground biomass by 16.4% in the greenhouse and generated 1.14 times more, but smaller stomata on leaves. Water stress in ECM inoculated seedlings also recovered faster from an experimental drought having significantly higher Fv/Fm value (P = 0.004) than uninoculated seedlings. Our study clearly indicates that ectomycorrhizal colonization can help chestnut trees in their early growth and stress tolerance and should be included into field plantings to minimize dependence on agricultural amendments.

Microbial Consortium Improved Growth and Performance of Teak (Tectona grandis L.f.) in Nursery and Field Trials

The response of teak (Tectona grandis L.f.) to the selected microbial consortium (Ambispora leptoticha + Azotobacter chroococcum + Trichoderma harzianum) was evaluated through large-scale nursery trials at three locations in Mandya district of Karnataka state, India. At each location, there were 500 inoculated and 500 uninoculated seedlings, totaling to the maintenance of 3000 seedlings at three locations. The growth performance was evaluated 180 days after planting. The increase in plant dry weight of inoculated seedlings was 97% compared to uninoculated plants. The seedlings inoculated with the microbial consortium under large-scale nursery trials were planted in the wasteland at three locations, and their growth was evaluated for nearly 6 years. The biovolume index of inoculated plants was 289% more than uninoculated plants 73 months after out-planting. It can be concluded that the selected microbial consortium is the best for inoculating teak in forest nurseries.

Microbial Biofertilizers and Soil Amendments Enhanced Tree Growth and Survival in a Barren Mined-out Area in Marinduque, Philippines

A bioremediation protocol was developed for rehabilitating mine tailing areas using microbial biofertilizers and soil amendments. The effects on the growth and survival of tree species in a three-decade barren mined-out area in Barangay Capayang, Mogpog, Marinduque, Philippines were also determined. Three concurrent field experiments were established in June 2016 using Pterocarpus indicus, Acacia mangium and Eucalyptus urophylla. Treatments for this study were uninoculated seedlings and seedlings inoculated with mycorrhiza (MYKORICH® for P. indicus) or Surigao isolate (for A. mangium and E. urophylla) with and without nitrogen-fixing bacteria. Aseptically germinated seedlings were inoculated when they were transplanted from seed boxes into individual polybags. After six months, the seedlings were planted in the mined-out area following Randomized Complete Block Design. The excavated soil were mixed with 1 kg vermicompost and 500 g lime before backfilling the 30 cm3 planting hole. After one month, 10 g NPK fertilizer and 5 g urea were applied on each seedling. Microbial-inoculated seedlings showed better growth performance with higher plant dry weight and microbial population compared to the uninoculated plants after 27 months. Hence, P. indicus, A. mangium, and E. urophylla inoculated with arbuscular mycorrhizal fungi and applied with lime, vermicompost, and basal inorganic fertilizer could be effective as reforestation species in barren mined-out areas.

Effect of the inoculation of plant growth-promoting rhizobacteria on the photosynthetic characteristics of Sambucus williamsii Hance container seedlings under drought stress

Plant growth-promoting rhizobacteria (PGPR) are beneficial bacteria that survive within the range of plant rhizosphere and can promote plant growth. The effects of PGPR in promoting plant growth, activating soil nutrients, reducing fertilizer application, and improving the resistance of plant inducible system have been widely investigated. However, few studies have investigated PGPR as elicitors of tolerance to abiotic stresses, especially drought stress. In this study, the effects of Acinetobacter calcoaceticus X128 on the photosynthetic rate (Pn), stomatal conductance (Gs), intracellular CO2 concentration (Ci), and total chlorophyll content [Chl(a+b)] of Sambucus williamsii Hance seedling leaves under moderate drought stress and drought-rewatering conditions were determined. Compared with those of uninoculated seedlings, the average Pn values during the entire drought stress of inoculated seedlings increased by 12.99%. As the drought duration was lengthened, Ci of uninoculated leaves continued to increase after rapidly declining, whereas Gs continuously decreased. Furthermore, their photosynthetic properties were simultaneously restricted by stomatal and non-stomatal factors. After X128 inoculation, Ci and Gs of S. williamsii Hance leaves continued to decrease, and their photosynthetic properties were mainly restricted by stomatal factors. At the end of the drought stress, water stress reduced [Chl(a + b)] of S. williamsii Hance leaves by 13.49%. However, X128 inoculation decreased this deficit to only 7.39%. After water supply was recovered, Pn, Gs, and [Chl(a+b)] in uninoculated leaves were reduced by 14.23%, 12.02%, and 5.86%, respectively, relative to those under well-watered conditions. However, Ci increased by 6.48%. Compared with those of uninoculated seedlings, Pn, Gs, and [Chl(a+b)] in X128-inoculated seedlings were increased by 9.83%, 9.30%, and 6.85%, respectively. Therefore, the inoculation of X128 under arid environments can mitigate the reduction of chlorophyll, delay the restriction caused by non-stomatal factors to Pn in plant leaves under water stress, and can be more conducive to the recovery of photosynthetic functions of leaves after water supply is recovered.

Mitigation of drought stress in wheat crop by drought tolerant endophytic bacterial isolates

Bacteria with potential to alleviate abiotic stresses in combination with plant growth promotion are extremely useful in sustainable agriculture. The present study involves the isolation and evaluation of 14 bacterial isolates obtained from four upland rice roots in varying concentrations of PEG-6000. Three potential isolates MKA2, MKA3 and MKA4 were selected for further experiments. A pot experiment was conducted with four treatments under well watered and drought conditions. Water deficit treatment was imposed by reducing the amount of water added to 25% of the field capacity. Twenty days post stress results showed that drought stress affected the physiological and biochemical parameters of uninoculated wheat seedlings. Inoculation with the three bacterial isolates diluted the adverse effects of drought on relative water content and membrane stability index, and thus improved the water use efficiency over the uninoculated control. Inoculation also improved the shoot and root biomass of wheat plants in comparison to uninoculated control treatment under drought stress. Increased levels of super oxide dismutase and catalase activities were observed in treated plants as compared to control plants thus protecting the plants from oxidative damage. Thus, these potent bacterial isolates could be used effectively to ameliorate plant water intake efficiency and improve sustainability in wheat crop under drought conditions.

Effects of an ectomycorrhizal fungus on the growth and physiology of Pinus sylvestris var. mongolica seedlings subjected to saline–alkali stress

This research investigates the mechanism of increased salinity tolerance of ectomycorrhizal fungi-inoculated P. sylvestris var. mongolica to provide a theoretical basis for the application of the fungus in saline soils. Growth effects due to inoculation of seedlings with Suillus luteus (a symbiotic ectomycorrhizal fungus), were determined in four kinds of saline–alkali soils. Growth and physiological indicators, including photosynthetic characteristics, plant height, biomass, photosynthetic pigments, catalase (CAT) and superoxide dismutase (SOD) enzyme levels, and malondialdehyde (MDA), an organic marker for oxidative stress, and soluble protein levels were determined. Mycorrhizal colonization rate decreased with increasing saline–alkalinity and growth of inoculated seedlings was significantly enhanced. Biomass and chlorophyll contents also increased significantly. SOD and CAT activities were higher than in non-inoculated seedlings. However, MDA content decreased in inoculated seedlings. Soluble protein content did not increase significantly. Inoculation with a symbiotic ectomycorrhizal fungus could enhance the saline–alkali tolerance of P. sylvestris var. mongolica. Growth and physiological performance of inoculated seedlings were significantly better than that of uninoculated seedlings. The results indicate that inoculated P. sylvestris var. mongolica seedlings may be useful in the improvement of saline–alkali lands.

Ph. D. Phytoremediation of Polluted Soil Using Some Ornamental Trees

This experiment was conducted at the Nursery of the Department of Floriculture, OrnamentalHorticulture and landscape Gardening - Faculty of Agriculture Shatby - University of Alexandria in twosuccessive seasons (2014-2015 and 2015-2016), to study the biological treatment of contaminated soil withsome heavy metals using some trees, namely;1. Acacia saligna (inoculated with Rhizobium sp.,).2. Casuarina glauca: which inoculated with Frankia.The treatments of the heavy metals (Cd - Zn - Pb) were: Control - 250 - 500 and 1000 mg/kg soil. Themost important results follows: The application of the high level of heavy metal (500- 1000 ppm) lead to decrease the growth parameters inboth seedlings. The negative effect of the pollutant was more significant in uninoculated seedlings withRhizobium and Frankia. Both of Cd and Pb had harmful impact on plant growth than that the Zn did. On the other hand, the effect ofthe Zn content was more harmful on C. glauca seedlings. Chlorophyll contents of the phyllodes were decreased as a result of application of heavy metal. The inoculated plants with Rhizobium and Frankia had displayed a significant increase in carotine contentunder high levels of heavy metals as it compared with uninoculated ones. Nitrogen and phosphorus contents were decreased in the phyllodes of inoculated seedlings with Rhizobiumand branchlets of Frankia with increasing of heavy metal level. The concentration of lead in the phyllodes was higher than that detected in the roots, particularly inuninoculated seedlings with Rhizobium, while for the Casuarina seedlings, both of Pb and Zn in the rootswas higher than that in the leaves.Advisors Committee Examiners CommitteeProf. Dr. Tarek Mahmoud El-kiey Prof. Dr. Emam Mohamed Saber NofalProf. Dr. Ahmed A. A. El-Settawy Prof. Dr. Essam Abd El-Rahman Koreish

Inoculation with Microbial Consortium Promotes Growth of Tomato and Capsicum Seedlings Raised in Pro Trays

Tomato and capsicum are important vegetable crops with wide usage in culinary. Inoculation of the planting medium with beneficial microbial consortium is an innovative approach to produce quality and healthy seedlings in horticulture. In the present study, inoculation of a selected microbial consortium consisting of Bacillus sonorensis + Funneliformis mosseae was added to the planting medium in pro trays to raise tomato and capsicum seedlings in a polyhouse. Considering growth parameters like germination rate, seedling length, seedling vigour index, stem girth, biovolume index, seedling dry weight; and microbial parameters like mycorrhizal root colonization and spore numbers, and population of B. sonorensis in the substrate, the ability of the consortium to improve seedling growth was assessed. The results revealed that the seedlings inoculated with the consortium showed significantly improved growth as compared to uninoculated seedlings. Based on the various growth and microbiological parameters studied, it was concluded that inoculation with consortium is beneficial for raising healthy, vigorously growing tomato and capsicum seedlings under polyhouse condition.

Psychrotolerant bacteria isolated from the leaf apoplast of cold-adapted wild plants improve the cold resistance of bean (Phaseolus vulgaris L.) under low temperature

We have isolated psychrotolerant bacteria from the leaf apoplast of cold-adapted wild plants and aimed to investigate their effect on the cold resistance of bean (Phaseolus vulgaris L.). Based on the findings of 16S rRNA gene sequence analysis, 20 isolates belonging to 5 bacteria species (Pseudomonas fragi, P. chloropaphis, P. fluorescens, P. proteolytica and Brevibacterium frigoritolerans) were identified in the leaf apoplastic fluid of Draba nemorosa, Galanthus gracilis, Colchicum speciousum, Scilla siberica, Erodium cicutarium, respectively. We have determined that 6 of the 20 isolates have exhibited ACC (1-aminocyclopropane-1-carboxylate) deaminase activity and secreted different extracellular proteins under cold condition (+4 °C) compared to normal growth condition (28 °C). The six isolates were then inoculated independently of each other to the leaves of 10-day-old bean seedlings growing under normal conditions (25/22 °C, 16/8 h photoperiod), and the inoculated and uninoculated (control) seedlings were transferred to cold (9/5 °C, 16/8 h photoperiod) for 3 days. The bacterial inoculations have decreased freezing injury, ice nucleating activity and lipid peroxidation content in parallel with the decrease of reactive oxygen species level such as O2.- and H2O2 in the inoculated seedlings compared to the control. In addition, the inoculations of the isolates have stimulated the activity of apoplastic antioxidant enzymes including superoxide dismutase, catalase, peroxidase, and glutathione reductase. The results show that the inoculations improve the cold resistance of bean seedlings and the psychrotolerant bacterial isolates can be evaluated within the group of plant growth promoting bacteria (PGPB) which can increase tolerance of cold-sensitive crops.

First Report of Phytophthora capsici Infection on Bell Peppers (Capsicum annuum L.) from Punjab, Pakistan

Bell pepper (Capsicum annuum L.) is one of the extensively cultivated vegetable crop in Punjab, Pakistan. During two years of field surveys, February-November 2016-17, damping off and blight symptoms were observed. Average seedling mortality was recorded as 18.7% while yield loss due to blight was estimated 32 to 41% at mature stages. Maximum blight infection was recorded from the areas frequently flooded with canal irrigation system. At early stages, lesions were noticed on stem portions at soil line level while at crop maturity stages blight symptoms were noted. Leaves were blanched and wilted while fruits were covered with white mold. Masses of sporangia were evident on and inside the infected fruits under humid conditions. A total of twelve isolates were recovered from infected root, stem and fruit portions on rye agar media (Caten and Jinks, 1968) incubated at 25oC under fluorescent light. Papillated sporangia were averaged 42 ± 2.6 X 27 ± 1.7 μm in size (range 27 - 52 × 23 - 36 μm). Oospores were produced on 20% V8 agar and were spherical 22 ± 1.4 μm in diameter (range 14 to 27 μm) while average pedicels length was recorded as 58 ± 12.5 μm (range 13 to 120 μm). These observations were similar to those described for P. capsici (Cocoa, 1988). DNA was extracted using Cetyl Trimethylammonium Bromide (CTAB) method and the internal transcribed spacer regions (ITS1 and ITS2) were amplified by polymerase chain reaction (White et al., 1990). The amplicons were purified and sequenced in both directions (GenBank Accession No. MF322868 and MF322869). BLAST analysis revealed these isolates showed 99% identity with ITS sequences of Phytophthora capsici (KM369964 and KU518782). Pathogenicity assay was performed on healthy bell pepper seedlings with five repeats. Soil was flooded with 20ml sporangial suspension (1 x 103 sporangia/ml) in pots containing seedlings while 5ml suspension was sprayed until run off on mature plants (Hyder et al., 2018). A set of uninoculated seedlings was used as control. Pots were kept in dew chamber for 10-20 days at 25±2 oC. Seedling mortality was observed five days after inoculation while at later stage plants develop brown-to-black stem lesions with white mycelial growth on leaves. These symptoms were identical to the P. capsici infections in field. Consistent re-isolations of P. capsici confirm its association with the disease. To our knowledge, this is the first report of Phytophthora blight on bell pepper from Pakistan

Arbuscular Mycorrhizal Symbiosis Modulates Antioxidant Response and Ion Distribution in Salt-Stressed Elaeagnus angustifolia Seedlings.

Elaeagnus angustifolia L. is a drought-resistant species. Arbuscular mycorrhizal symbiosis is considered to be a bio-ameliorator of saline soils that can improve salinity tolerance in plants. The present study investigated the effects of inoculation with the arbuscular mycorrhizal fungus Rhizophagus irregularis on the biomass, antioxidant enzyme activities, and root, stem, and leaf ion accumulation of E. angustifolia seedlings grown during salt stress conditions. Salt-stressed mycorrhizal seedlings produced greater root, stem, and leaf biomass than the uninoculated stressed seedlings. In addition, the seedlings colonized by R. irregularis showed notably higher activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) in the leaves of the mycorrhizal seedlings in response to salinity compared to those of the non-mycorrhizal seedlings. Mycorrhizal seedlings not only significantly increased their ability to acquire K+, Ca2+, and Mg2+, but also maintained higher K+:Na+ ratios in the leaves and lower Ca2+:Mg2+ ratios than non-mycorrhizal seedlings during salt stress. These results suggest that the salt tolerance of E. angustifolia seedlings could be enhanced by R. irregularis. The arbuscular mycorrhizal symbiosis could be a promising method to restore and utilize salt-alkaline land in northern China.

Bioaccumulation of cadmium by Enterobacter sp. and enhancement of rice seedling growth under cadmium stress

Bacteria-mediated plant growth promotion and bioremediation of heavy metal containing soil is a widely accepted eco-friendly method. The present study is aimed to screen out cadmium resistant bacterial strain from metal contaminated rice rhizosphere and evaluate its effects on the growth of rice seedlings under cadmium stress. Among four different isolates (designated as S1, S2, S3 and S5), the S2 isolate was screened on the basis of different PGP traits and multi heavy metal resistance (minimum inhibitory concentration for cadmium, lead and arsenic were 3500, 2500 and 1050 µg/ml respectively). The selected S2 strain has ability to produce ACC deaminase (236.11 ng α-keto-butyrate/mg protein/h), IAA (726 µg/ml), solubilize phosphate (73.56 ppm) and fix nitrogen (4.4 µg of nitrogen fixed/h/mg protein). The selected strain was identified as Enterobacter sp. on the basis of phenotypic characterization, MALDI-TOF MS analysis of ribosomal proteins, FAME analysis and 16 S rDNA sequence homology. The high cadmium removal efficiency (> 95%) of this strain from the growth medium was measured by Atomic Absorption Spectrophotometer and it was due to intracellular cadmium accumulation evidenced by SEM-EDX-TEM-EDX study. SEM analysis also revealed no distortion of surface morphology of this strain even grown in the presence of high cadmium concentration (3000 µg/ml). Inoculation of this strain with rice seedlings significantly enhanced various morphological, biochemical characters of seedling growth compared with un-inoculated seedlings under Cd stress. The strain also exhibited alleviation of cadmium-induced oxidative stress, reduction of stress ethylene and decreased the accumulation of cadmium in seedlings as well that conferred cadmium tolerance to the plant. Thus the S2 strain could be considered as a potent heavy metal resistant PGPR applicable in heavy metal contaminated agricultural soil for bioremediation and plant growth promotion as well. Main findingA cadmium resistant plant growth promoting Enterobacter sp. was isolated that accumulated cadmium evidenced by SEM-TEM-EDX study. It reduced Cd uptake and enhanced growth in rice seedlings.

Transcriptional response of oil palm (Elaeis guineensis Jacq.) inoculated simultaneously with both Ganoderma boninense and Trichoderma harzianum

Application of beneficial microbes offers an environmentally friendly alternative for mitigation of basal stem rot (BSR) disease in oil palm. However, the biocontrol mechanisms of Trichoderma against the pathogenic Ganoderma spp. which cause BSR are largely unknown at the molecular level. To identify the transcripts involved during induced systemic resistance (ISR), we analyzed the root transcriptomes of oil palm seedlings inoculated simultaneously with both G. boninense and T. harzianum, and un-inoculated oil palm seedlings, as well as those that were inoculated with either pathogenic or beneficial fungi. Our analyses revealed that the biocontrol mechanisms of T. harzianum against G. boninense involve modulation of genes related to biosynthesis of phytohormones (ethylene, MeJA and MeSA), antioxidant (l-ascorbate and myo-inositol) and unique secondary metabolites such as momilactone, cell wall metabolisms, and detoxification of phytotoxic compounds; in addition to its role as a biofertilizer which improves nutritional status of host plant. The outcomes of this study have fueled our understanding on the biocontrol mechanisms involving T. harizianum against G. boninense infection in oil palm roots.

The Plant Growth-Promoting Fungus (PGPF) Alternaria sp. A13 Markedly Enhances Salvia miltiorrhiza Root Growth and Active Ingredient Accumulation under Greenhouse and Field Conditions.

Plant growth-promoting fungi (PGPF) have attracted considerable interest as bio-fertilisers due to their multiple beneficial effects on plant quantity and quality and their positive relationship with the ecological environment. Advancements in the development of PGPF for crops and economic plant cultivation applications have been achieved, but such improvements for the use of PGPF with popular medicinal herbs, such as Salvia miltiorrhiza, are rare. In this study, we collected S. miltiorrhiza specimens inhabiting wild, semi-wild, farmland and pot-cultured areas in the Henan province of China and isolated endophytes from the roots, shoots and leaves of these samples. Twenty-eight strains of the dominant genus Alternaria were identified and selected as candidate PGPF. Under greenhouse conditions, Alternaria sp. A13 simultaneously enhanced the dry root biomass and secondary metabolite accumulation of S. miltiorrhiza as the optimal PGPF of the 28 candidate isolates. To further assess the interaction between S. miltiorrhiza and Alternaria sp. A13, the effects on seedlings growth, active ingredient accumulation, and the activity of key enzymes for effective biosynthetic pathways were investigated over a period of six months under field conditions. Compared to uninoculated seedlings, S. miltiorrhiza seedlings colonised by Alternaria sp. A13 showed significant increment of 140% in fresh weight, 138% in dry weight, and enhancement in the contents of total phenolic acid, lithospermic acids A and B (LAA and LAB, respectively) of 210%, 128% and 213%, respectively. Examination of the related enzyme activities showed that the elicitation effect of A13 on LAB accumulation correlated with cinnamic acid 4-hydroxylase (C4H) activity in the phenylpropanoid pathway under field conditions. Our results confirmed that Alternaria sp. A13 not only contributes to the stimulation of S. miltiorrhiza root growth, but also boosts the secondary metabolism, thus demonstrating its application potential as a bio-fertiliser for S. miltiorrhiza cultivation, especially in areas outside of its native growth regions.

A rare temperate terrestrial orchid selects similar Tulasnella taxa in ex situ and in situ environments

Mycorrhizal symbiosis in orchids is unique in that fungal presence is considered a requirement for germination as well as for further development. Additionally, orchid fungal associations can exhibit high specificity in nature. Yet, an important ecological question remains unanswered: ‘With which orchid mycorrhizal fungi (OMF) do un-inoculated orchid seedlings form symbiosis when cultured ex situ?’ Simultaneously, it is asserted that orchid conservation efforts involving ex situ plant culture should exclusively utilize natural symbionts of the respective orchid taxa. We present a first comparison of OMF communities within the roots of asymbiotically cultured plants of the rare orchid Platanthera chapmanii grown ex situ (ES), and those occurring naturally in situ (IS). Nuclear ribosomal internal transcribed spacer (nrITS) barcoding region was used to identify peloton forming OMF from roots collected between 2012 and 2014 from both growing environments. Our 114 sequences clustered into 11 operational taxonomic units (OTUs) belonging to four closely related clades of the fungal family Tulasnellaceae. Shannon–Wiener (H) and Simpson diversity (D) indices were similar (p = 0.81 for both) for ES and IS OMF communities. Beta diversity comparisons also showed similarity between ES and IS treatments based on weighted (p = 0.10) and unweighted (p = 0.20) Bray–Curtis dissimilarity matrices. Bayesian and Maximum Likelihood (ML) phylograms clustered ES and IS derived fungal OTUs into the same clades. Our data suggest that P. chapmanii: (1) forms symbiosis with taxonomically similar fungi in ex situ culture and in its native soil, and (2) exhibits a narrow phylogenetic breadth of mycorrhizal fungal OTUs within the Tulasnellaceae.

Coinoculation of bioinoculants improve Acacia auriculiformis seedling growth and quality in a tropical Alfisol soil

We conducted a study to find out if arbuscular mycorrhizal (AM) fungi (Acaulospora scrobiculata, Scutellospora calospora) and phosphate solubilizing bacteria (PSB, Paenibacillus polymyxa) inoculation either individually or in combinations can improve Acacia auriculiformis seedling growth, uptake of nutrients and quality in a phosphorus deficient tropical Alfisol. The seedlings were assessed for various growth and nutrient uptake parameters after 60 days of treatment. Inoculation with P. polymyxa stimulated mycorrhizal formation. Seedling height, stem girth, taproot length, number of leaves and leaf area, plant dry matter production, nodulation, and nodular dry weight were significantly higher for seedlings that were either dual inoculated or triple inoculated compared to individual inoculation of AM fungi or PSB, and uninoculated seedlings. Dual and triple application of AM fungi and PSB also significantly improved the nutrient contents of shoots and roots and nutrient uptake efficiencies. The calculated seedling quality indexes of the AM fungi and PSB inoculated seedling were 25–208% higher than uninoculated seedlings. These findings show that A. auriculiformis seedlings when dual inoculated or triple inoculated performed better than seedlings inoculated with the microbes individually and compared with uninoculated control seedlings. We conclude that bioinoculation is important for the production of high-quality A. auriculiformis seedlings in tree nurseries for planting in nutrient deficient soils.

Ectomycorrhizal Fungi and Mineral Interactions in the Rhizosphere of Scots and Red Pine Seedlings

Ectomycorrhizal fungi and associated bacteria play a key role in plant-driven mineral weathering and uptake of mineral-derived nutrients in the rhizosphere. The goal of this study was to investigate the physical and chemical characteristics of bacteria-fungi-mineral interactions in biofilms of Scots and red pine rhizospheres. In three experiments, seedlings were grown in columns containing silica sand amended with biotite and calcium-feldspar, and inoculated with pure cultures of ectomycorrhizal fungi or a soil slurry. Uninoculated seedlings and unplanted abiotic columns served as controls. After nine months, the columns were destructively sampled and the minerals were analyzed using scanning electron and atomic force microscopy. Element release rates were determined from cation concentrations of input and output waters, soil exchange sites, and plant biomass, then normalized to geometric surface area of minerals in each column. The results revealed that various ectomycorrhizal fungal species stimulate silicate dissolution, and biofilm formation occurred at low levels, but direct surface attachment and etching by fungal hyphae was a minor contributor to the overall cation release from the minerals in comparison to other environmental conditions such as water applications (rain events), which varied among the experiments. This research highlights the importance of experimental design details for future exploration of these relationships.