iForest - Biogeosciences and Forestry
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Use of overburden waste for London plane (Platanus × acerifolia) growth: the role of plant growth promoting microbial consortia

iForest - Biogeosciences and Forestry, Volume 10, Issue 4, Pages 692-699 (2017)
doi: https://doi.org/10.3832/ifor2135-010
Published: Jul 17, 2017 - Copyright © 2017 SISEF

Research Articles

Overburden waste dumps represent a huge threat to environmental quality. The reduction of their negative impact can be achieved by vegetation cover establishment. Usually, this action is complicated due to site-specific characteristics, such as nutrient deficiency, elevated metal concentration, low pH value, lack of moisture and lack of organic matter. Establishment of vegetation can be facilitated by inoculation with plant growth promoting bacteria (PGPB) which improve the physicochemical and biological properties of degraded substrates and make them more hospitable for plants. In this study we selected several strains based on the ability to produce ammonia, indole-3-acetic acid, siderophores and lytic enzymes, and to solubilize inorganic phosphates. This selection resulted in microbial consortia consisting of Serratia liquefaciens Z-I ARV, Ensifer adhaerens 10_ ARV, Bacillus amyloliquefaciens D5 ARV and Pseudomonas putida P1 ARV. The effects of PGPB consortia on one-year-old London plane (Platanus × acerifolia [Aiton] Willd.) seedlings replanted into overburden waste from Kolubara Mine Basin were examined. After seven months, inoculated seedlings were 32% higher with 45% wider root collar diameter and over 80% higher total dry biomass compared to uninoculated seedlings grown in Kolubara’s overburden. Inoculation resulted in higher amounts of total soluble proteins, higher chlorophyll and epidermal flavonoids content and higher total antioxidative capacity in the leaves. This study represents a successful search for effective PGPB strains and shows that microbial consortia have an important role in enhancing the growth of seedlings in nutrient deficient and degraded substrates such as overburden waste from open-pit coal mines. Positive response of London plane seedlings suggest that inoculation may help widening the opus of species for reforestation of post mining areas and speed up natural succession processes and recovery of degraded landscapes.

Plant Growth Promoting Bacteria, London Plane, Overburden Waste, Revegetation

  Introduction 

Soils can be disturbed by a wide range of factors concerning unfavorable agricultural management, industry, mining activities, etc. Surface mining exerts long-term negative impact on the environment, destroying large areas of natural landscapes. During coal exploitation geological layers above and around the ore body are disturbed, and piled up in mixtures forming overburden deposits. The disposal of overburden is made non-selectively, resulting in new relief forms ([37]). The image of open-pits destructive character is visible only 50 km southwest of Belgrade (Serbia) at Kolubara Mine Basin (Lazarevac district, Serbia). Currently, at this location, mining activity occupies over 5730 ha while overburden waste dumps cover 3395 ha. Even though large areas of overburden dumps are an environmental issue, causing erosion, water and air pollution, recultivation has been carried out on only 882 ha (Report on the state of environment in Branch MB Kolubara, Lazarevac - 2015).

Revegetation of overburden waste dumps is a worldwide problem, and establishment of vegetation cover on such foundations is complicated due to a numerous problems such as nutrient deficiency, elevated metal concentration, low pH value, lack of moisture, soil forming materials and of organic matter, high heterogeneity of substrate, disturbed soil hydrology and topography ([37], [20]). At impoverished mine sites, enrichment with organic matter (cover crops, mulch, compost, hay) and encouragement of symbiotic relationships between plants and soil microbes makes a difference between life and death ([46]). In addition to already mentioned approaches, inoculation with plant growth promoting bacteria (PGPB) is emerging as a promising technique ([36], [20]).

PGPB reside in the rhizosphere, root surface, and plant inner tissues ([15]) and stimulate plant growth through a variety of mechanisms. PGPB directly affect plant growth by improving nutrient assimilation through fixation of atmospheric nitrogen, siderophores production, solubilization of phosphorus, and/or other unavailable forms of nutrients ([15]). Production and/or suppression of growth regulating hormones (auxins, gibberellins, cytokinins) is another effective direct mechanism for plant growth enhancement. Indirect mechanisms include antagonism against phytopathogens, niche competition, and increased disease resistance ([15]).

Plant growth promoting bacteria improve physicochemical and biological properties of poor, degraded substrates and make them more suitable for plants ([16], [20]) with simultaneous increase of plant survival and seedling quality, especially in soils with weak microbial activity ([10], [12]). Positive outcomes of PGPB application are enhancement of seedlings emergence, faster plant growth, higher biomass production ([36]), increase of root length, and branching, increased leaf area, and chlorophyll content, and higher resistance to abiotic stresses ([15]) as well as to pests or diseases ([7]).

After more than a century of research, beneficial effects of PGPB are now exploited in agriculture, horticulture, forestry and environmental restoration ([25]). Studying the interactions between PGPB and angiosperms/gymnosperms started during 1980s and 1990s ([10]); however, up to now, the opus of tested species cover a very few genera, such as Pinus sp., Tsuga sp., Pseudotsuga sp., Quercus sp., Eucalyptus sp. etc. ([38], [36]).

In previous attempts, Kolubara Mine landscapes were reforested with Black pine, Scots pine, European larch, European ash, Small-leaved lime, and Black locust ([20]). Recultivation was mainly conducted with pioneer species, even though some of them were not the most desirable for our climate conditions (e.g., Black locust). Through their beneficial activity on soil and plants, PGPB may help widening the opus of species for reforestation of overburden areas by providing the space for more demanding ones. This assumption was tested in the present study and London plane (Platanus × acerifolia [Aiton] Willd.) was used as test plant. The ornamental values of plane trees make them a valuable part of urban landscapes all over the world. The members of Platanus spp. are well known as street trees but also are suitable for phytoremediation purposes and reforestation of post-mined land ([44], [19]). Interestingly, even though London plane is one of the most frequent species in the public open space, so far there have been no studies dedicated to London plane - PGPB interactions.

Our starting hypothesis was that the overburden waste enriched with PGPB may represent a suitable substrate for London plane growth. For this purpose the search for the most effective PGPB strains was conducted. The final confirmation of PGPB beneficial influence was obtained through in vivo experiments. Monitoring the influence on London plane growth and performances in overburden waste confirmed the success of the search.

  Material and methods 

Collection of bacterial isolates

Forty four isolates of soil bacteria from the Laboratory for Microbial Ecology, Faculty of Agriculture, University of Belgrade (Serbia) represented the starting point of the experiment and were tested for the presence of plant growth promoting features.

Plant growth-promoting (PGP) features

Ammonia (NH3) production was tested by growing isolates in 10 ml peptone water. After 48-72 h at 28 °C, Nessler’s reagent (0.5 ml) was added in each tube. Development of brown to yellow color was a positive test for ammonia production ([6]).

Assay for indoleacetic acid (IAA) production was conducted using the colorimetric method ([32]). The isolates were grown in 10 ml of minimal salt media supplemented with 100µg ml-1 of l-tryptophan (Sigma Aldrich, USA). Absorption was read after 48 h at 530 nm with a spectrophotometer (T70 UV/VIS Spectromer, PG Instruments LTD, UK). The amount of IAA was determined using a calibration curve of indole-3-acetic acid (Sigma Aldrich, USA) in the 1-100 µg ml-1 range.

Siderophore production was detected on the Chrome azurol S (Fluka, USA) agar medium ([1]). Chrome azurol S agar plates were spot inoculated with test organisms and incubated at 28 °C for 48-72 h. Development of yellow-orange halo zones around colonies was considered as positive result for siderophore production.

Phosphate solubilizing activity was tested on National Botanical Research Institute’s phosphate growth medium (NBRIP - [30]). Strains were incubated for 14 days at 30 °C, and afterwards the presence of halo zones around colonies were used to indicate their phosphate solubilization capability. P solubilization index was calculated using the following formula (eqn. 1):

\begin{equation} SI = {\frac{ \oslash_{colony} + \oslash_{halozone}} { \oslash_{colony}}} \end{equation}

Qualitative determination of lipase, N-acetyl-β-glucosaminidase and β-glucosidase was performed by API ZYM kits according to the manufacturer’s protocol (Bio Mereux, France). The API strips were inoculated with 24-h-old cultures, and incubated at 30 °C for 4 h. The evaluation of the activity was carried out by comparing the colored reaction with the manufacturer’s color chart. Protease production was determined using skim milk agar ([9]). The agar plates were spot inoculated with test organisms and incubated at 30 °C for 5 days. The presence of a clear zone around the colonies indicated protease activity. The presence of cellulase was determined using carboxymethyl cellulose (CMC) agar method ([3]). The agar plates were spot inoculated and incubated at 30 °C for 48 h. Plates were flooded with 0.1% congo red solution and were destained with 1M NaCl solution after 10 minutes. The appearance of clearing zones around colonies indicated cellulase activity.

Hydrogen cyanide (HCN) production was determined using nutrient broth amended with glycine (4.4 g l-1). Bacteria were streaked on agar plate. Whatman filter paper no. 1 soaked in 2% sodium carbonate in 0.5% picric acid solution was placed at the top of the plate. Plates were sealed with parafilm and incubated at 28 °C for 4 days. Development of orange to red color indicated HCN production.

Molecular identification of isolates

Isolates that showed the most prominent plant growth promoting activities were molecularly identified by sequencing the gyrB gene. Genomic DNA was prepared by using ZR Soil Microbe DNA MiniPrep (Zymo Research, USA). The amplification of gyrB gene was performed with a thermal cycler (Kyratec, Australia, Model: SC300T) using the primer sets presented in Tab. 1.

Tab. 1 - Primer pairs used for PCR amplification of gyrB gene fragments (R=A or G; Y=C or T; M=A or C; N=any).

Primers Sequence (5’-3’) Isolate
gyr-320 (fw) TAARTTYGAYGAYAACTCYTAYAAAGT Z-I ARV ([11])
rgyr-1260 (rv) CMCCYTCCACCARGTAMAGTTC
UP-1E (fw) CAGGAAACAGCTATGACCAYGSNGGNGGNAARTTYRA 10_ARV, D5 ARV ([50])
modUP2r (rv) TGTAAAACGACGGCCAGTCCRTCNACRTCNGCRTCNGTCAT
UP-1E (fw) CAGGAAACAGCTATGACCAYGSNGGNGGNAARTTYRA P1 ARV ([50])
APrU (rv) TGTAAAACGACGGCCAGTGCNGGRTCYTTYTCYTGRCA

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The reaction mixtures (50 µl) contained 0.2 mM of each dNTP (Kapa Biosystems, UK), 1 µM of each primer, 0.5 U Robust HotStart DNA Polymerase (Kapa Biosystems, UK) and 20 ng of DNA template. PCR reactions were performed as previously described by Dauga ([11]) and Yamamoto et al. ([50]). PCR products were purified with QIAquick PCR Purification Kit (Qiagen, Germany), and sequenced with the primers presented in Tab. 2.

Tab. 2 - Primer pairs used for sequencing (R=A or G; Y=C or T; M=A or C).

Primers Sequence (5’-3’) Isolate
gyr-320 (fw) TAARTTYGAYGAYAACTCYTAYAAAGT Z-I ARV
rgyr-1260 (rv) CMCCYTCCACCARGTAMAGTTC
M13R-pUC (fw) CAGGAAACAGCTATGAC 10_ARV, D5 ARV, P1 ARV
M13-FP (rv) TGTAAAACGACGGCCAGT

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PCR products were sequenced on ABI 3730XL Sequencer (Macrogen Inc., Seoul, Korea) in both directions. Alignment of obtained sequences was performed using the Clustal W 2.0 algorithm and MEGA5 software. The BLAST database of National Centre for Biotechnology Information (NCBI - ⇒ http:/­/­www.­ncbi.­nlm.­nih.­gov) was used to compare the sequence of bacteria with those of known bacterial species in the existing database. Sequences were deposited in the GenBank database (Accession Numbers: KT265088, KT265086, KT265087, and KT265089, for Z-I ARV, 10_ARV, D5 ARV, and P1 ARV strains, respectively).

Substrates and plant material

Substrates used for plant cultivation were Floradur® Plant Universal (FloraGard, Germany) and overburden waste from Kolubara Basin (Serbia). Floradur® Plant Universal contained: 50% white peat, 30% black peat, and 20% compost, with pH 5.8. Analyses of overburden were performed at the beginning of the experiment, before planting. Samples were taken from 20 different spots according to standard soil sampling principles. Obtained amount was homogenized and successive splitting by cone and quarter technique was applied. Overburden was composed of 43.2% clay, 52.7% silt, and 4.1% sand (determined with soil texture triangle according to [40]). Previous analyses of overburden waste showed neutral pH value, low content of nitrogen, humus (0.15%), and of organic carbon ([20]). Prior to use, overburden waste was air-dried, ground, and sieved through a 2 mm diameter sieve.

One-year-old bare root seedlings of Platanus x acerifolia Aiton (Willd.) with mean height of 19.55 cm and mean collar diameter of 0.29 cm were obtained from the Nursery of forest and ornamental plants Vikumak (Idoš, Serbia).

Microbial consortia inoculum preparation

Inocula were prepared from four separately propagated isolates. Three bacterial strains (Serratia liquefaciens Z-I ARV, Bacillus amyloliquefaciens D5 ARV and Psedomonas putida P1 ARV) were grown in nutrient broth aerobically at 28 ± 2 °C / 48 h / 100 rpm (Biosan, Latvia). Ensifer adhaerens 10_ARV was grown in Fjodorov medium ([2]) at 28 ± 2 °C / 72 h / 100 rpm. The bacterial suspensions were centrifuged at 6000 × g for 10 minutes (5804 R, Eppendorf, Germany) and diluted in sterile distilled water to adjust bacterial cell density to 108 CFU mL-1. Bacterial strain-specific inocula were mixed together to form a consortia inoculum in the 1:1:1:2 ratio (Serratia liquefaciens Z-I ARV: Bacillus amyloliquefaciens D5 ARV: Psedomonas putida P1 ARV: Ensifer adhaerens 10_ARV).

In vivo trials

The experiment was based on three treatments: (a) seedlings replanted into commercial substrate, Floradur® Plant Universal (FS); (b) seedlings replanted into overburden (O); (c) seedlings replanted into overburden and inoculated with PGPB consortia (OI). All seedlings were placed in 2.5 dm3 volume of substrate in 3 dm3 polyethylene bags during the period of dormancy. Inoculation was conducted two times, at the beginning of the growing season (March) and 12 weeks later. Each seedling in the OI treatment received 100 ml of inoculum prepared as described above. Seedlings in the other two treatments (FS and O) received 100 ml of distilled water. Plants were grown until the beginning of October. The temperature conditions in Belgrade during the experiment are presented in Tab. 3. The trial was performed outdoors at a location of the Faculty of Agriculture in Belgrade (Serbia) and was arranged as a completely randomized split-plot design (n = 20 seedlings per treatment).

Tab. 3 - Daily maximum and daily minimum temperature range during the outdoor experiment (data for Belgrade, Serbia, obtained from the Hydrometeorological Service of Serbian Republic).

Period Daily maximum
temperature range (°C)
Daily minimum
temperature range (°C)
February (dormant season) -0.3 → 23.4 -6.4 → 8.8
Spring (March, April, May) 8.4 → 29.7 1.5 → 17.8
Summer (June, July, August) 18.0 → 34.8 11.0 → 22.9
September 15.8 → 28.3 8.0 → 20.0

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Plant growth measurements and leaf analyses

Seedling height and root collar diameter were recorded two times. The first measurement was conducted 18 weeks after the first inoculation (July) and the second in October. At the end of the experimental period (October), shoot and root dry weights were also recorded. Plants from each treatment were grouped and dried at 65 °C until constant weight.

For biochemical analyses, fully developed, green and vital mid-shoot leaves without visible signs of senescence were sampled in the beginning of October (harvest period) and ground in liquid nitrogen. Soluble proteins were extracted in 100 mM potassium-phosphate buffer (pH 6.5) with 0.1% Triton X-100 (w/v), 5% insoluble polyvinylpyrrolidone (PVP) and 1 mM phenylmethylsulfonyl fluoride (PMSF), according to Vidović et al. ([47]).

Following centrifugation at 16.000 × g (5415 R, Eppendorf, Germany) for 10 min at 4 °C, protein content in the soluble fraction was determined using bovine serum albumin as standard ([4]). For determination of total soluble phenolics content, leaf samples were homogenized in liquid nitrogen and extracted in methanol with 0.1% HCl ([47]). Following centrifugation, the supernatants were separated and the content of total soluble phenolics was determined using the Folin-Ciocalteu reagent, as described by Morina et al. ([29]). The concentrations of phenolics in methanolic extracts were calculated using gallic acid as a standard ([43]). Total antioxidative capacity of methanolic extracts was measured using 2.2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) ABTS assay, as described by Morina et al. ([29]). For simultaneous in vivo assessment of total leaf chlorophyll and epidermal flavonoids content, optical leaf-clip meter Dualex4Scientific (Force-A, Orsay, France) was used, as described in Cerović et al. ([8]).

Statistical analysis

Data were analyzed by two-way analysis of variance (ANOVA) followed by LSD tests (p < 0.05) and Independent Two Sample t-test. The analyses were conducted using the software package SPSS v. 22 (SPSS Inc., Chicago, IL, USA).

  Results and discussion 

Overburden waste dumps represent huge areas of unproductive land with erosion susceptibility and hazardous influence on soil, water, and air quality. These problems are usually solved through vegetation development ([37]).

One of the promising ways for raising the effectiveness of overburden revitalization is the application of PGPB, and this has been successfully achieved in the present study. After series of biochemical tests, isolates with the most prominent PGP features were identified as Serratia liquefaciens Z-I ARV (KT265088), Ensifer adhaerens 10_ARV (KT265086), Bacillus amyloliquefaciens D5 ARV (KT265087) and Psedomonas putida P1 ARV (KT265089). These four strains were selected from the collection of 44 soil isolates which were tested for the presence of the most frequent PGPB features (Tab. 4).

Tab. 4 - Plant growth promoting attributes of isolated strains. (NH3): ammonia; (IAA): indoleacetic acid; (Sid): siderophores; (PSI): phosphate solubilization index; (+) presence of the feature; (-) absence of the feature. (*): selected strains.

Isolates Production PSI
NH3 IAA
(µg ml-1)
Sid
3N ARV - 1.7 ± 0.3 + -
6N ARV + 0.4 ± 0.1 - -
7N ARV + 0.8 ± 0.1 - 2.33 ± 0.45
8N ARV + 0.8 ± 0.2 - -
15N ARV + 0.4 ± 0.1 - -
19 D ARV + 0.5 ± 0.02 - -
Z-I1 ARV + 9.6 ± 0.2 + 2.25 ± 0.32
Z-I ARV* + 8.4 ± 0.3 + 2.60 ± 0.30
Z-H ARV + 0.5 ± 0.1 - -
CSP ARV - 2.6 ± 0.3 - -
G1SP ARV + 2.0 ± 0.2 - -
GSP ARV - 1.8 ± 0.3 - -
2TSB ARV + 0.8 ± 0.1 - -
O3 ARV + 1.1 ± 0.2 - -
2T ARV + 10.5 ± 0.5 - -
F ARV + 0.4 ± 0.1 - -
333 ARV - 26.4 ± 0.5 + 2.11 ± 0.22
10_ARV* + 44.5 ± 0.5 + 2.33 ± 0.30
NV1 ARV + 4.0 ± 0.2 - 2.11 ± 0.25
NV2 ARV + 5.6 ± 0.4 + -
NV3 ARV + 3.8 ± 0.4 + 2.14 ± 0.15
NV4 ARV + 4.0 ± 0.3 + -
NV5 ARV + 8.0 ± 0.4 - 1.50 ± 0.15
NV6 ARV + 2.4 ± 0.3 - -
NV7 ARV + 1.1 ± 0.2 - -
4 ARV + 1.4 ± 0.2 - -
8 ARV + 0.4 ± 0.1 - 2.22 ± 0.25
D1 ARV + 0.8 ± 0.1 - -
D2 ARV + 1.1 ± 0.15 - -
D3 ARV + 0.5 ± 0.1 - 2.27 ± 0.28
D5 ARV* + 1.5 ± 0.3 + -
D6 ARV + 1.8 ± 0.2 - -
P1 ARV* + 1.2 ± 0.1 + 2.70 ± 0.25
T1 ARV - 1.4 ± 0.1 - -
T6 ARV + 4.0 ± 0.2 + -
T10 ARV - 1.4 ± 0.3 - 2.50 ± 0.30

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The results of biochemical test have shown that NH3 was produced by 33 isolates (75%), whereas 36 isolates (82%) were characterized as IAA producers. Such results were expected since these two features are usual among beneficial soil bacteria ([14]). The results of IAA production distinguished Ensifer adhaerens 10_ARV which produced considerably higher amounts compared to others (44.5 µg ml-1). Similar findings on E. adhaerens have been noted by Kaur et al. ([22]). Indole-3-acetic acid is the most common form of auxins ([14]) which affect plant cell division, extension, and differentiation, increase the rate of root development and nodulation, initiate lateral and adventitious root formation and stimulate seed and tuber germination. Metabolic processes such as photosynthesis, biosynthesis of various metabolites, and resistance to stressful conditions are modulated by auxins ([15]). Accordingly, IAA indirectly increases water and nutrient supplies leading to higher root exudation and biomass production ([14], [15]).

The production of siderophores was expressed by 11 isolates. This characteristic of PGPB is very important since the amount of available iron in soil is limited ([14]). To survive with a restricted supply of iron, bacteria produce low-molecular weight compounds (siderophores) with high affinity for Fe+3 ([14]). PGPB siderophores raise Fe supply to plants and lower down the Fe amounts available for plant pathogens ([14]). In addition, siderophores form stable complexes with Al, Cd, Cu, Ga, In, Pb and Zn and are important for reducing the level of plant stress caused by high concentration of heavy metals ([14]).

Solubilization of phosphates is another crucial feature of PGPB which provides more H2PO4- and HPO42- for plants ([14]). Slightly higher number of isolates showed positive result in phosphate solubilization testing (27%) compared to siderophores production. Serratia liquefaciens Z-I ARV and Pseudomonas putida P1 ARV expressed the highest P solubilization ability. Ensifer adhaerens 10_ARV was also capable to solubilize inorganic phosphates (Tab. 4). This characteristic was already recorded among other representatives of these three bacterial species ([18], [42], [22]).

Platanus × acerifolia is very tolerant to numerous abiotic stresses such as limited root space, unfavourable soil conditions, drought, air pollution, urban climate ([28]) and exhibits high accumulation capacity of heavy metals ([17], [19]). Those features make plane trees suitable for urban, surface mine areas ([44]) and phytoremediation activities ([19]). On the other side, a number of pests and pathogens can threaten this genus causing considerable damage. Some of the most destructive are canker stain caused by Ceratocystis fimbriata Ell.et. Halsted f.sp. platani, anthracnose by Apiognomonia veneta (Sacc. et Speg.), Hohn, powdery mildew by Microsphera platani (Howe) and trunk rot by Phytophthora cinnamomi Rands ([34]). Considering the prevalence of plane trees in urban areas all over the world it is important to develop suitable measures to prevent or restrict the pathogens. However, up to now, the application of phytosanitary measures, chemical treatments and biological control measures were not particularly efficient ([31], [35]). A genetic approach with the aim of selecting resistant plane genotypes is believed to be an effective way of disease control ([35]). Breeding programs with American sycamore (Platanus occidentalis L.) and oriental plane tree (Platanus orientalis L.) resulted in cultivars “Columbia” and “Liberty”, which are highly resistant to Apiognomonia veneta (Sacc. et Speg.), Hohn ([24]). In addition, Vigouroux & Olivier ([48]) and Pilotti et al. ([33]) produced genotypes resistant to Ceratocystis fimbriata Ell.et. Halsted f.sp. platan.

However, the selection for resistant genotypes is very extensive while large-scale usage of a small number of clones can be detrimental for plane trees biodiversity ([35]). This emphasizes the need for more intensive selection activities and new approaches which may include biological control. PGPB with biocontrol features already represent an effective measure against numerous plant pathogens ([15], [7]). The mechanisms employed by biocontrol bacteria are diverse and include antibiotics, siderophores and lytic enzymes production which are all effective against Botrytis cinerea, Fusarium spp., Phytophthora spp., Rhizoctonia solani, Pythium ultimum, etc. ([15]). PGPB are also capable to activate induced systemic resistance (ISR), and prepare plant defense mechanisms for potential pathogen attack. ISR is not pathogen-specific and it can effectively suppress diseases caused by different plant pathogens ([15]). All those mechanisms secure wide spectra of PGPB action. Combination of resistant genotypes or genotypes with certain level of resistance with PGPB may represent a new approach in reducing the incidence or severity of plane diseases. Also, PGPB with biocontrol function can be applied through soil introduction or foliar application which is effective for suppression of foliar diseases ([26]). Genetic manipulation with potentially effective biocontrol agent may result in superior biocontrol strains, and offer even more promising results ([14]).

In our study, the biocontrol potential of the isolates was estimated trough production of several lytic enzymes (lipase, N-acetyl-β-glucosaminidase, β-glucosidase, protease, cellulase), which are capable of damaging fungal cell-walls ([15], [7]). These tests revealed that only ten isolates produce substances which are known to have antifungal activity (Tab. 5). Serratia liquefaciens Z-I ARV, Pseudomonas putida P1 ARV and Bacillus amyloliquefaciens D5 ARV were capable to produce lytic enzymes. These species have already been recognized as effective biocontrol agents ([49], [7], [41]). The only isolate that stood up after screening of PGP direct mechanisms but did not express any indirect mechanism was Ensifer adhaerens 10_ARV.

Tab. 5 - The presence of lytic enzymes. (Naβ): N-acetyl-β-glucosaminidase; (β-glu): β-glucosidase; (HCN): hydrogen cyanide; (+) presence of the feature; (-) absence of the feature; (*): selected strains.

Isolates Lytic enzymes
lipase Naβ β-glu protease cellulase
7N ARV - - - + -
Z-I ARV* + + + + -
G1SP ARV - + - - -
333 ARV + + + + -
NV4 ARV - - - + -
NV5 ARV - - - + -
7 ARV + - - - -
D5 ARV* - - - + +
D6 ARV + - - - -
P1 ARV* - + + - -

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New tendencies in bacterial inoculum application emphasize the ecological advantages of mixed populations over single strain inocula ([14]). After series of in vitro tests, inoculum consisting of Serratia liquefaciens Z-I ARV, Ensifer adhaerens 10_ARV, Bacillus amyloliquefaciens D5 ARV and Pseudomonas putida P1 ARV was used for in vivo experiments aimed at confirming the PGPB potential of selected isolates under field conditions. S. liquefaciens Z-I ARV was selected based on high P solubilization index and the ability to perform all four direct mechanisms and produce several lytic enzymes. E. adhaerens 10_ARV showed the highest IAA production. B. amyloliquefaciens D5 ARV showed ability to use several direct mechanisms and was the only isolate that produced cellulase. P. putida P1 ARV stood up with the highest P solubilization index among tested isolates.

Plant-bacteria interactions are under strong influence of abiotic factors and indigenous microflora. Those two factors are the most common cause of PGPB failure in uncontrolled environment ([20]) and in vivo testing is crucial for proper assessment of PGPB potential.

The effects of applied treatments on height and root collar diameter are presented in Tab. 6. At the start of the experiment, the seedlings had similar features (height and root collar diameter). After 12 weeks inoculated seedlings (OI) had similar height to those grown in the commercial substrate (FS), but were significantly higher compared to uninoculated overburden seedlings (O). At the end of the experiment the FS seedlings showed the highest growth increment, followed by OI seedlings, while O treatment seedlings were the smallest. Comparison of overburden seedlings revealed positive effect of inoculation on height increment.

Tab. 6 - The height and root collar diameter of Platanus × acerifolia seedlings. (OI): seedlings re-planted into overburden and inoculated with PGPB consortia; (O): seedlings re-planted into overburden; (FS): seedlings re-planted into commercial substrate (Floradur® Plant Universal). The first measurement was in July, and the second measurement was in October. Mean values and standard errors (n=20) are reported. Values in the same column with different letters differ significantly (p<0.05) according to the LSD test.

Treatment Seedling height (cm) Root collar diameter (cm)
Start 1st meas. 2nd meas. Start 1st meas. 2nd meas.
OI 19.98 ± 4.31a 37.00 ± 5.11b 42.60 ± 4.88b 0.30 ± 0.05a 0.57 ± 0.07b 0.68 ± 0.07b
O 19.33 ± 3.55a 28.43 ± 5.03a 32.33 ± 4.65a 0.27 ± 0.07a 0.40 ± 0.08a 0.47 ± 0.08a
FS 19.35 ± 4.12a 39.55 ± 6.05b 47.43 ± 5.98c 0.30 ± 0.04a 0.71 ± 0.15c 0.77 ± 0.13c
LSD0.05 3.105 0.056

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Measurements of root collar diameter conducted in July put the treatments in the following order, FS>OI>O and this trend was maintained until the end of the experiment. In addition, positive effect of inoculation on seedlings width was observed when comparing OI and O treatments.

At the end of the experiment, seedlings were uprooted; roots and shoots were separated and dried until constant weight. Tab. 7 shows roots and shoots dry biomass of Platanus × acerifolia seedlings. The highest biomass production was noted in FS treatment. The OI treatment was intermediate as it yielded significantly higher root and shoot biomass than the O treatment, suggesting that inoculation had a positive influence on seedlings biomass production.

Tab. 7 - Roots and shoots dry biomasses of Platanus × acerifolia seedlings. (OI): seedlings re-planted into overburden and inoculated with PGPB consortia; (O): seedlings re-planted into overburden; (FS): seedlings re-planted into commercial substrate (Floradur® Plant Universal); RDB: root dry biomass; SDB: shoot dry biomass. Mean values and standard errors (n=20) are reported. Values in the same column with different letters differ significantly (p<0.05) according to the LSD test.

Treatment RDB (g) SDB (g)
OI 2.35 ± 0.94b 4.34 ± 0.65b
O 1.23 ± 0.91a 2.47 ± 0.98a
FS 3.73 ± 2.69c 7.28 ± 3.46c
LSD0.05 1.091 1.336

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Comparison of inoculated and uninoculated overburden seedlings revealed positive effects of PGPB consortia on plant performances (Tab. 6, Tab. 7). Egamberdiyeva ([13]) claimed that poor substrates are more suitable for exhibiting the full potential of PGPB, while in rich substrates plants are already well supplied. In our in vivo test one third of the seedlings were grown in commercial substrate with the aim of comparing performances of inoculated seedlings to those grown in nutrient sufficient substrate. At the end of the experiment, even though applied PGPB consortia enhanced London plane growth, their effects were not strong enough to parry seedlings grown in commercial substrate, suggesting that inoculation can compensate the lack of nutrients, but to a certain extent (Tab. 6, Tab. 7).

Seedlings grown in FS were 10% higher with 12% wider root collar diameter, 40% higher shoot dry biomass and 37% higher root dry biomass in comparison to OI seedlings. On the other hand, comparison of uninoculated and inoculated overburden seedlings showed that the presence of PGPB caused 32% increase in seedling height, 45% wider root collar diameter, 76% increase of shoot dry biomass and 91% of root dry biomass (Tab. 6, Tab. 7). Similar results were reported by Karlidag et al. ([21]) where inoculation with Bacillus sp. and Microbacterium sp. induced 30% increase of apple shoot length. Rodriguez-Barrueco et al. ([38]) reported 90% increment of oak seedlings biomass after inoculation with Azospirillum brasilense, while Mesorhizobium sp. increased White birch biomass by 60% ([45]). Moreover, we have previously reported that inoculation with Azotobacter chroococcum and several Bacillus sp. strains resulted with 34% higher root dry biomass of Scots pine and 23% of Norway spruce seedlings ([20]).

The data on PGPB effects on plant morphology are voluminous, and recent studies are more interested in physiological aspects of inoculation. In this manner efforts are directed towards revealing PGPB influences on mineral content of plants, chlorophyll content, and accumulation of secondary metabolites ([39]). At the end of our experiment, fitness condition of Platanus × acerifolia inoculated and uninoculated overburden seedlings were estimated through the content of total soluble proteins, total soluble phenolics, total chlorophyll, and several other parameters (Tab. 8). A significant influence of applied consortia on total soluble proteins content, chlorophyll content, total antioxidative capacity, and epidermal flavonoids content has been revealed. On the other hand, inoculation did not affect the total soluble phenolics content. Increase of leaf chlorophyll content, amount of total proteins, together with higher shoot growth and total dry matter, indicate an enhanced nutrient assimilation, as similarly observed by Mia et al. ([27]). Increased total antioxidative capacity in OI treatment compared to O seedlings may be of great importance for stress amelioration ([39]), especially considering that the seedlings are intended for harsh environments, such as overburden waste dumps. Successful adaptation and growth of plants in inhospitable environments highly depends on species choice, seedling quality ([20]), and the ability to recover the damaged root system shortly after replanting. The most critical moment is replanting from nurseries to final place. At this point, the presence of PGPB can be of great help considering their influence on transplant shock mitigation ([23]). Rapid development of new roots, increase of root growth, length and weight are commonly reported responses in trees to PGP bacteria inoculations ([21], [45]). These effects has also been confirmed in the present study where inoculation caused 91% increase of root dry biomass (Tab. 7), presumably by modulating endogenous plant mechanisms which regulate root development ([36]).

Tab. 8 - Fitness condition of Platanus × acerifolia overburden seedlings. (OI): seedlings re-planted into overburden and inoculated with PGPB consortia; (O): seedlings re-planted into overburden. Mean values and standard errors (n=12) are reported. Different letters in the same column indicate significant differences (p<0.05) after Independent Two Sample t-test.

Treatment Σ proteins
(mg gFW-1)
Σ phenolics µmol
gallic acid eq.
(gFW-1)
Σ chlorophyll
(mg cm-2)
Σ antioxidative
capacity µmol
ascorbate eq.
(gFW-1)
epidermal
flavonoids
(g cm-2)
OI 1.41 ± 0.32b 119.03 ± 9.69a 16.6 ± 1.06b 10.09 ± 2.01b 1.13 ± 0.161b
O 0.94 ± 0.11a 105.88 ± 13.07a 13.9 ± 0.94a 4.93 ± 1.10a 0.38 ± 0.286a

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Literature data emphasize the validity of PGPB inoculation in revegetation projects of anthropogenically devastated areas. This has been further confirmed by the results obtained in the present study. All benefits that PGPB inoculation provides to London plane seedlings may be of great help in alleviating overburden waste dumps challenges. Also, such effects may be crucial for trees in urban areas considering their constant exposure to numerous stresses ([5]). The main issue is finding the suitable bacteria strains that will be capable to express their beneficial effects in uncontrolled conditions. Considering the obtained results, our search has been successful, and the presence of the PGPB consortia in overburden waste raised its suitability for plant growth. Positive response of London plane seedlings suggests that inoculation may help widening the opus of species for reforestation of post-mining areas. This may be the proper measure that opens the door for more demanding species and speeds up natural succession processes and recovery of degraded landscapes.

  Conclusion 

PGPB used in our study were selected based on their PGP mechanisms. In vivo experiments with PGPB consortia confirmed their plant growth promoting nature through stimulating effects on London plane height, root collar diameter, total biomass production, and fitness. This study justifies the PGPB inoculation as a proper technique in mitigation of overburden waste dumps issues.

  Acknowledgments 

This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Grant No. TR 31080.

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Authors’ Affiliation

(1)
Vera Karličić
Danka Radić
Jelena Jovičić-Petrović
Blažo Lalević
Vera Raičević
Faculty of Agriculture, University of Belgrade, Nemanjina 6, Zemun (Serbia)
(2)
Filis Morina
Institute for Multidisciplinary Research, University of Belgrade, Kneza Višeslava 1, Belgrade (Serbia)
(3)
Vesna Golubović Curguz
Faculty of Forestry, University of Belgrade, Kneza Višeslava 1, Belgrade (Serbia)

Corresponding author

 

Citation

Karličić V, Radić D, Jovičić-Petrović J, Lalević B, Morina F, Curguz VGć, Raičević V (2017). Use of overburden waste for London plane (Platanus × acerifolia) growth: the role of plant growth promoting microbial consortia. iForest 10: 692-699. - doi: 10.3832/ifor2135-010

Academic Editor

Claudia Cocozza

Paper history

Received: Jun 14, 2016
Accepted: Apr 12, 2017

First online: Jul 17, 2017
Publication Date: Aug 31, 2017
Publication Time: 3.20 months

© SISEF - The Italian Society of Silviculture and Forest Ecology 2017

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