The Effect of Biochars and Endophytic Bacteria on Growth and Root Rot Disease Incidence of Fusarium Infested Narrow-Leafed Lupin (Lupinus angustifolius L.).

The effects of biochar on plant growth vary depending on the applied biochar type, study site environmental conditions, microbial species, and plant–microbial interactions. The objectives of the present study were therefore to assess 1) the response of growth parameters of lupin and root disease incidence to the application of three biochar types in a loamy sandy soil, and 2) the role of endophytic bacteria in biological control of root rot disease incidence in lupin after the amendment of soil with different biochar types. As biochar types we tested (i) hydrochar (HTC) from maize silage, (ii) pyrolysis char from maize (MBC), and (iii) pyrolysis char from wood (WBC) at three different concentrations (1%, 2%, and 3% of char as soil amendments). There were no significant effects in lupin shoot and root growth in soils amended with WBC at any of the concentrations. MBC did not affect plant growth except for root dry weight at 2% MBC. HTC char at 2% concentration, significantly increased the root dry weight of lupin by 54–75%, and shoot dry weight by 21–25%. Lupin plants grown in soil amended with 2% and 3% WBC and MBC chars showed 40–50% and 10–20% disease symptoms, respectively. Plants grown in soil without biochar and with HTC char were healthy, and no disease incidence occurred. Pseudomonas putida L2 and Stenotrophomonas pavanii L8 isolates demonstrated a disease reduction compared to un-inoculated plants under MBC and WBC amended soil that was infested with Fusarium solani.


Introduction
Biochar is a solid by-product of biomass produced by pyrolysis or by high temperatures under limited supply of or in the complete absence of air [1]. It is considered to be a tool for carbon sequestration and to improve soil and crop health [2][3][4]. The amendment of soil with biochar was reported to enhance plant growth and yields, such as of pepper and tomato [5], soybean [6], maize [7], or wheat [8]. Moreover, biochar holds the potential as a suitable carrier of microbial inoculants to improve plant growth [9]. The biochar application improved soil cation exchange capacity [10], water holding

Plant Growth Experiment
The pot experiment was conducted under greenhouse conditions at the Leibniz Center for Agricultural Landscape Research (ZALF), Germany. Three different biochar types, WBC, MBC, and HTC, were used as soil amendments. Pots (d = 0.16 m, v = 3016 cm 3 ) were filled with 1000 g of air-dried soil mixed with crushed char (particle size <3 mm) at 1%, 2%, and 3% (w/v) concentration. The following four treatments were set up: i) plants grown in soil without WBC, MBC, or HTC biochar, ii) plants grown in soil amended with WBC, iii) plants grown in soil amended with MBC, iv) plants grown in soil amended with HTC.
Each treatment included four replicated pots, arranged in a randomized complete block design with three replications (total of 12 plants), and three char concentrations (1%, 2%, and 3% w/v). Plants were grown for 30 days at a temperature of 24 • C/16 • C (day/night) and humidity of 50-60%. At harvest, the lupin root system was carefully removed, soil adhering to the roots was washed and examined for root rot symptoms. The dry weights of roots and shoots of healthy plants were determined.

Isolation of Fungi from Diseased Plants
The plants grown under biochar amendment (WBC) showing Fusarium root rot symptoms were collected, surface disinfected by dipping into 70% ethanol and then 1% NaOCl for 5 min, and were washed with sterile distilled water three times. The sterilized plants were cut into small pieces and placed on the surface of potato dextrose agar (PDA) (BD, Difco Laboratories, Detroit, USA) supplemented with chloramphenicol (Oxoid, UK) (150 ppm). The plates were kept in an incubator for five days, then fungal colonies with different morphological characteristics were isolated, and purified. The identification of fungi at the genus level was carried out by using macroscopic and microscopic examinations depending on the colony color, shape, hyphae, conidia, conidiophores, and arrangement of spores. The isolated fungal isolates belong to Mucor spp., Alternaria spp., and Fusarium spp.
The plant pathogenicity tests were conducted with two Fusarium spp. and Alternaria spp. isolates in order to select pathogenic fungi that cause lupin root rot disease. A single culture was prepared from each isolate. The pathogenicity of these isolates was tested by sowing lupin seeds in pots filled with sterile vermiculite and sand (1/1) mixed with spores of the pathogen (7.0 × 10 5 spores/kg soil) and grown for six weeks. At harvest, the diseased plants were selected, and a piece of the root system was placed on a PDA plate and re-isolated. The fungal isolates were grown on sterile filter paper placed on PDA agar and then ground in liquid nitrogen. The Nucleon Phytopure kit (Amersham Biosciences GmbH, Freiburg, Germany) was used to isolate DNA. The fragments of mtSSU rDNA were amplified using MS1 and MS2 primers [35], and the sequences of the fragments were compared with those in the GenBank using the BLAST algorithm. The isolate showed 99.68% similarity with the mtSSU rDNA sequence from F. solani f. sp. glycines 91-10-1-G (GenBank Acc.N. AF125009.1) and was identified as F. solani.

Isolation of Endophytic Bacteria from Lupin Roots and Characterization
Lupin grown under field conditions at the Experimental Station of the Leibniz Center for Agricultural Landscape Research (ZALF), Germany was collected for the isolation of endophytic bacteria. Four fresh plants were separated from the shoots and roots and washed, and then surface sterilized by immersion in 70% ethanol and 5% sodium hypochlorite.
One gram of root was macerated in a mortar and mixed with 9 mL sterile buffered saline (PBS) (20 mM sodium phosphate, 150 mM NaCl, pH 7.4). The supernatant was diluted (10 1 -10 5 ) and spread on Tryptic Soy Agar (TSA) (BD, Difco Laboratories, Detroit, USA). The plates were incubated for four days at 28 • C, and morphologically different colonies were picked up from the plates and purified.

DNA Isolation
For DNA extraction, the heat treatment method [36] was used. The small aliquots of the colonies were transferred into 2-mL Eppendorf tubes with 1.5 mL of sterile MQ-water and were mixed with a Biosan B-1 Vortex for 10 sec. The tubes were incubated at 90 • C for 20 min in a Dry Block Heater (IKA Works) and centrifuged at 12,000 rpm for 5 min. The DNA-containing supernatant was taken and stored at −20 • C. The presence of DNA was checked by horizontal gel electrophoresis (0.8% agarose) and was quantified using NanoDrop™ One (Thermo Fisher Scientific, Inc. Waltham, MA).

Restriction Fragment Length Polymorphism (RFLP) Analysis
To determine the difference of the isolates similar in their color, shape, and size, we conducted an RFLP analysis of 16S rRNA products as described by Jinneman et al. [37]. The fragments of digested RNA were checked via gel electrophoresis (1% agarose gel). After the electrophoresis, the gel was visualized using a digital gel imaging system (Gel-Doc XR TM+, Bio-Rad) to identify identical isolates to reduce the number of strains to be sequenced.

Sequencing and Phylogenetic Analysis
Before being sequenced, the PCR products were purified with the USB ® ExoSAP-IT ® PCR Product Cleanup Kit (Affymetrix, USB ® Products, USA) according to the manufacturer's protocol. Sequencing was performed using an ABI PRISM BigDye 3.1 Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) by the manufacturer's protocol. Received data were analyzed and corrected using Chromas (v. 2.6.5) software. Corrected sequences were merged manually using EMBOSS Explorer (http://emboss.bioinformatics.nl/). The sequences were identified using the basic local alignment search tool (BLAST), and comparisons were made with the GenBank nucleotide data bank from the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/). All sequences were multiply aligned using Clustal× 2.1 software, and a FASTA format file was used to construct the phylogenetic tree. The evolutionary history was inferred using the neighbor-joining method [38]. The optimal tree with the sum of branch length = 0.76327423 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches [39]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method [40] and are in the units of the number of base substitutions per site. The analysis involved 19 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1281 positions in the final dataset. Evolutionary analyses were conducted in MEGA6 [41].
The 16S rRNA sequences isolated from the endophytic bacteria of white lupin were deposited into GenBank under the accession numbers: MH636785-MH636793.

Plant Growth Traits
Several plant growth-promoting traits of endophytic bacteria were determined. Indole 3-acetic acid (IAA) production was studied following the method described in Bano and Musarrat [42]. The production of chitinase and protease activity was determined as described in Malleswari and Bagyanarayan [43]. The cellulose-degrading capacity of the bacterial isolates was analyzed as described by Gupta et al. [44]. The antagonistic capabilities of bacterial isolates against the pathogenic fungi F. oxysporum, F. solani, Fusarium culmorum, Verticillum dahlia, and R. solani were evaluated on potato dextrose agar (PDA) media as described by Egamberdieva et al. [6].

Biological Control of Lupin Root Rot
The biological control of lupin root rot caused by F. solani under different biochar soil amendments using selected endophytes was determined in a pot experiment. For soil infestation, a suspension of F. solani grown in Czapek-Dox medium for four days was filtrated with sterile glass wool, centrifuged, and diluted to 10 6 spores mL −1 . The resulting suspension with fungal spores was mixed with soil (10 6 spores kg −1 soil). Two biochar types MBC and WBC were mixed with the soil at 2% (w/w) as follows: Each of the treatments contained four groups of twenty-four plants and were arranged in a randomized block design. For plant inoculation, bacterial isolates were grown in Tryptic soy broth (TSB) medium for two days, centrifuged, washed in PBS, and re-suspended with a bacterial density of 10 8 CFU/mL. The seedling roots were dipped into bacterial suspension prior to planting into soil. The plants were grown at a temperature range of 24-28 • C day and 18-20 • C night under greenhouse conditions for 34 days. At harvest, root rot symptoms of washed plants were determined as indicated by browning and lesions.

Plant Growth Experiment with Endophytic Bacteria and Biochar
The plant growth chamber experiments with nine treatments were set up as described above. The lupin seeds were surface-sterilized using 70% ethanol and 10% v/v NaOCl. Afterward, the seeds were washed five times with sterile distilled water and transferred to paper tissue for germination in a dark room at 25 • C for 3-4 days. The bacterial strains P. putida L2 and S. pavanii L8 were grown in TSB medium for two days, and the culture suspension was re-suspended in PBS with a final bacterial density of 10 8 CFU mL −1 . Germinated seeds were dipped into the bacterial suspensions and transferred to pots. Three seeds were sown in each pot, and, after one week, the seedlings were thinned to one plant per pot. Each treatment included four pots and was arranged in a randomized complete block design with three replications (total of 12 plants). Plants were grown for 30 days at a temperature of 24 • C/16 • C (day/night) and humidity of 50-60%. At harvest, the roots and shoots were oven-dried at 70 • C for 48 h, and dry weight was determined.

Survival of Endophytic Bacterial Strains
The rifampicin (200 µg/mL) resistant mutants of the wild-type strain were obtained following the method of Glandorf et al. [45] and were used for the colonization studies. Mutants of P. putida L2 and S. pavanii L8 that were marked with antibiotic resistance were obtained by plating the parental strain onto TSA agar plates containing increasing concentrations of 50, 100, 150, and 200, µg/mL rifampicin. After incubation, isolates were selected based on similarities in colony morphology and growth rate with the parent strain and were re-cultured on medium containing 200 µg/mL rifampicin to ensure stability of the antibiotic resistance.
Plants were grown in pots as described above. Lupin seedlings were coated with bacteria by dipping the seedlings in bacterial suspensions that resulted in 10 8 CFU mL −1 seeds. Plants were grown for 30 days at a temperature of 24 • C/16 • C (day/night) and at a humidity of 50-60%. At harvest, the adhering soil was removed from plant roots, and 1 g roots was shaken in 9 mL sterile PBS. The resulting suspensions were evaluated for colony forming units (CFUs) according to the dilution-plate method in TSA agar with the addition of 200 µg/mL rifampicin. After incubation for 2-3 days at 28 • C the re-isolated, rifampicin-resistant strains were identified for their colony characteristics [46].

Statistical Analysis
Data were tested for statistical significance using the analysis of variance package included in Microsoft Excel 2010. Comparisons were performed using Student's t-test. Mean comparisons were conducted using the least significant difference (LSD) test (P = 0.05).

Response of Lupin to the Biochar Types
The response of narrow-leafed lupin to the type of biochar was studied at 1%, 2%, and 3% concentrations in pot experiments. The shoot and root biomass of lupin were not significantly affected by WBC soil amendments at 1-3% ( Figure 1A). Correspondingly, the root and shoot growth of lupin under MBC char was not affected, but only root dry weight was increased at 2% MBC amendment ( Figure 1B). However, the root dry weight of lupin was significantly increased by 54-75%, and the shoot dry weight was increased by up to 21-25% by HTC char amendment at 2% concentration, respectively ( Figure 1C).
The plants grown in soils amended with 2% and 3% w/v WBC and MBC chars showed disease symptoms of 40-50% and 10-20%, respectively ( Figure 2). Plants grown in soil without biochar and in soil with HTC char were healthy, and no disease incidence was found. The plants grown in soils amended with 2% and 3% w/v WBC and MBC chars showed disease symptoms of 40%-50% and 10%-20%, respectively ( Figure 2). Plants grown in soil without biochar and in soil with HTC char were healthy, and no disease incidence was found.  The plants grown in soils amended with 2% and 3% w/v WBC and MBC chars showed disease symptoms of 40%-50% and 10%-20%, respectively ( Figure 2). Plants grown in soil without biochar and in soil with HTC char were healthy, and no disease incidence was found.

Fungal and Bacterial Isolates
The selected fungal isolate showed root rot symptoms after plant pathogenicity tests and was identified as F. solani. The biological control capability of endophytic bacteria against root rot of lupin caused by F. solani was examined.
A total of 25 bacterial isolates were isolated from plant tissues of lupin. The RFLP analysis was conducted to eliminate similar isolates, and as a result, only nine isolates were selected and identified using the BLAST algorithm and compared with related strains from NCBI GenBank ( Table 2). Table 2. Sequence similarities of endophytic bacteria isolated from the roots of lupin grown under

Fungal and Bacterial Isolates
The selected fungal isolate showed root rot symptoms after plant pathogenicity tests and was identified as F. solani. The biological control capability of endophytic bacteria against root rot of lupin caused by F. solani was examined. A total of 25 bacterial isolates were isolated from plant tissues of lupin. The RFLP analysis was conducted to eliminate similar isolates, and as a result, only nine isolates were selected and identified using the BLAST algorithm and compared with related strains from NCBI GenBank (Table 2). All strains were identified using BLAST and compared with similar strains from the NCBI GenBank. The 16S rRNA sequences similarities of endophytic bacteria isolated from plant tissue of lupin with sequences from GenBank are shown in Table 2. All strains were identical with a similarity of 98.72-99.25% to those registered in GenBank. The isolates were identified as Bacillus pumilus L1, P. putida L2, Enterobacter cloacae L3, Pseudomonas koreensis L4, Pseudomonas thivervalensis L5, Pseudomonas corrugata L6, Pseudarthrobacter oxydans L7, S. pavanii L8, and Cellulosimicrobium cellulans L9 (Table 2, Figure 3). A phylogenetic tree was constructed showing the closest relatives of the isolates (Figure 3). All strains were identified using BLAST and compared with similar strains from the NCBI GenBank. The 16S rRNA sequences similarities of endophytic bacteria isolated from plant tissue of lupin with sequences from GenBank are shown in Table 2. All strains were identical with a similarity of 98.72%-99.25% to those registered in GenBank. The isolates were identified as Bacillus pumilus L1, P. putida L2, Enterobacter cloacae L3, Pseudomonas koreensis L4, Pseudomonas thivervalensis L5, Pseudomonas corrugata L6, Pseudarthrobacter oxydans L7, S. pavanii L8, and Cellulosimicrobium cellulans L9 (Table 2, Figure 3). A phylogenetic tree was constructed showing the closest relatives of the isolates ( Figure 3).
All nine endophytes were screened for multiple plant growth-promoting traits. Most of the bacterial isolates exhibited one or more plant growth-promoting activities (Table 3). Among nine isolates only two isolates P. putida L2 and S. pavanii L8 were able to produce IAA (5.6 and 4.7 µg mL -1 ), were able to produce one or more cell wall degrading enzymes, and were highly effective in vitro against the F. oxysporum and F. solani (Table 3). HCN and siderophores were produced only by P. putida L2.   All nine endophytes were screened for multiple plant growth-promoting traits. Most of the bacterial isolates exhibited one or more plant growth-promoting activities (Table 3). Among nine isolates only two isolates P. putida L2 and S. pavanii L8 were able to produce IAA (5.6 and 4.7 µg mL −1 ), were able to produce one or more cell wall degrading enzymes, and were highly effective in vitro against the F. oxysporum and F. solani (Table 3). HCN and siderophores were produced only by P. putida L2.

Biological Control of Lupin Root Rot under Biochar Amended Soil
The two selected isolates, P. putida L2 and S. pavanii L8, were chosen to evaluate their capability to suppress lupin root rot caused by F. solani. The portion of diseased plants grown in soil infested with the pathogen was 13%. The disease incidence was increased by 18% under MBC amended soil and by 45% under WBC soil amendment (Figure 4). The bacterial isolates, P. putida L2 and S. pavanii L8, demonstrated a disease reduction up to 9-12% under MBC amended soil compared to control plants without biochar (13%) and un-inoculated plants grown under MBC (18%) ( Figure 5). The number of pathogen-infected plants was reduced up to 20% and 28% by bacterial isolates P. putida L2 and S. pavanii L8 under WBC soil amendment, whereas un-inoculated plants grown under WBC char showed a disease incidence up to 45%.

Plant Growth, Nodulation, and Bacterial Survival in the Root System
The endophytic bacteria that showed biocontrol ability to control the root rot of lupin were evaluated for their plant growth-promoting activities under the amendment of two different biochar types. The responses of lupin to the two biochar types were different, being higher in soil amended with MBC compared to those in soil amended with WBC ( Figure 6). The shoot and root biomass of un-inoculated lupin grown under MBC char were increased by 6% and 14% compared to control plants, respectively. Bacterial inoculants P. putida L2 and S. pavanii L8 increased lupin root and shoot growth in soil without biochar amendment by 17-39% and 15-37%, respectively ( Figure 6). However, there were no significant effects on the shoot growth of inoculated plants grown in soil amended with MBC and WBC chars. The root biomass of lupin was increased by inoculation of plants with P. putida L2 in MBC and WBC amended soil ( Figure 6). Compared with plants grown in soil amended with MBC and WBC chars, the weight of shoots and roots was not affected by bacterial inoculation with S. pavanii L8. and by 45% under WBC soil amendment (Figure 4). The bacterial isolates, P. putida L2 and S. pavanii L8, demonstrated a disease reduction up to 9%-12% under MBC amended soil compared to control plants without biochar (13%) and un-inoculated plants grown under MBC (18%) ( Figure 5). The number of pathogen-infected plants was reduced up to 20% and 28% by bacterial isolates P. putida L2 and S. pavanii L8 under WBC soil amendment, whereas un-inoculated plants grown under WBC char showed a disease incidence up to 45%.

Plant Growth, Nodulation, and Bacterial Survival in the Root System
The endophytic bacteria that showed biocontrol ability to control the root rot of lupin were evaluated for their plant growth-promoting activities under the amendment of two different biochar types. The responses of lupin to the two biochar types were different, being higher in soil amended with MBC compared to those in soil amended with WBC ( Figure 6). The shoot and root biomass of un-inoculated lupin grown under MBC char were increased by 6% and 14% compared to control plants, respectively. Bacterial inoculants P. putida L2 and S. pavanii L8 increased lupin root and shoot growth in soil without biochar amendment by 17%-39% and 15%-37%, respectively ( Figure 6). However, there were no significant effects on the shoot growth of inoculated plants grown in soil amended with MBC and WBC chars. The root biomass of lupin was increased by inoculation of plants with P. putida L2 in MBC and WBC amended soil ( Figure 6). Compared with plants grown in soil amended with MBC and WBC chars, the weight of shoots and roots was not affected by bacterial inoculation with S. pavanii L8. Both endophytic bacteria P. putida (L2) and S. pavanii (L8) improved the symbiotic performance of lupin with its rhizobia under soil amended with MBC and WBC chars. The nodule numbers of lupin inoculated with P. putida L2 and S. pavanii L8 were 6.8 and 4.6 grown in soil without biochar, while under WBC and MBC biochar amendment nodule numbers increased to 9.0 and 10.0 per plant, respectively (Figure 7). However, there were no significant effects on the shoot growth of inoculated plants grown in soil amended with MBC and WBC chars. The root biomass of lupin was increased by inoculation of plants with P. putida L2 in MBC and WBC amended soil ( Figure 6). Compared with plants grown in soil amended with MBC and WBC chars, the weight of shoots and roots was not affected by bacterial inoculation with S. pavanii L8. Both endophytic bacteria P. putida (L2) and S. pavanii (L8) improved the symbiotic performance of lupin with its rhizobia under soil amended with MBC and WBC chars. The nodule numbers of lupin inoculated with P. putida L2 and S. pavanii L8 were 6.8 and 4.6 grown in soil without biochar, while under WBC and MBC biochar amendment nodule numbers increased to 9.0 and 10.0 per plant, respectively (Figure 7).
Rifampicin-resistant mutants of bacterial isolates P. putida L2 and S. pavanii L8 were tested for their survival in the root of lupin grown without and with biochar amendment. The results showed that both bacterial isolates were able to survive in the rhizosphere of lupin. The root colonization in the rhizosphere of lupin was 2.1 × 10 3 ± 1.4 with P. putida L2, and 3.2 × 10 3 ± 1.9 with S. pavanii L8 in soil without biochar amendment. The CFUs of bacterial isolates were slightly higher in WBC and MBC amended soils, being 3.2 × 10 3 ± 0.7 and 4.2 × 10 3 ± 1.2 (CFU/g of fresh root) with P. putida L2 and 3.9 × 10 3 ± 1.6 and 3.8 (CFU/g of fresh root) with S. pavanii L8, respectively. Both endophytic bacteria P. putida (L2) and S. pavanii (L8) improved the symbiotic performance of lupin with its rhizobia under soil amended with MBC and WBC chars. The nodule numbers of lupin inoculated with P. putida L2 and S. pavanii L8 were 6.8 and 4.6 grown in soil without biochar, while under WBC and MBC biochar amendment nodule numbers increased to 9.0 and 10.0 per plant, respectively (Figure 7).
Rifampicin-resistant mutants of bacterial isolates P. putida L2 and S. pavanii L8 were tested for their survival in the root of lupin grown without and with biochar amendment. The results showed that both bacterial isolates were able to survive in the rhizosphere of lupin. The root colonization in the rhizosphere of lupin was 2.1 × 10 3 ± 1.4 with P. putida L2, and 3.2 × 10 3 ± 1.9 with S. pavanii L8 in soil without biochar amendment. The CFUs of bacterial isolates were slightly higher in WBC and MBC amended soils, being 3.2 × 10 3 ± 0.7 and 4.2 × 10 3 ± 1.2 (CFU/g of fresh root) with P. putida L2 and 3.9 × 10 3 ± 1.6 and 3.8 (CFU/g of fresh root) with S. pavanii L8, respectively.

Discussion
Biochar amendments have been reported to elicit complex responses in plants [4,6,15], including plant growth stimulation or suppression of several soil-borne diseases [47,48]. However, the beneficial effect depends on the type of biochar feedstock, the technique of preparation (e.g., pyrolysis or carbonization), and finally, concentration [15,16]. Overall, our experiment showed that the shoot and root biomass of lupin responded differently to types of biochar and biochar concentrations, i.e., MBC char produced more benefits to lupin growth as compared to WBC char. Reibe et al. [33] reported similar observation for MBC char amendments increasing dry biomass of wheat as compared to WBC char or plants grown without any biochar application. Joseph et al. [49] reported that the interaction of biochar with environmental conditions is another crucial requirement for revealing contrasting effects, which might depend on the physicochemical properties of biochars. For example, Butnan et al. [25] observed reduced plant growth on a sandy Ultisol amended with eucalyptus wood-derived biochar produced by pyrolysis (800 °C), whereas biochar produced at a lower temperature (350 °C) provided higher benefits. Shen et al. [50] reported that the addition of biochar from willow woodchips increased plant growth of Lotus pedunculatus cv barsille, whereas pine-based biochar did not show any positive effect on plant growth. Rifampicin-resistant mutants of bacterial isolates P. putida L2 and S. pavanii L8 were tested for their survival in the root of lupin grown without and with biochar amendment. The results showed that both bacterial isolates were able to survive in the rhizosphere of lupin. The root colonization in the rhizosphere of lupin was 2.1 × 10 3 ± 1.4 with P. putida L2, and 3.2 × 10 3 ± 1.9 with S. pavanii L8 in soil without biochar amendment. The CFUs of bacterial isolates were slightly higher in WBC and MBC amended soils, being 3.2 × 10 3 ± 0.7 and 4.2 × 10 3 ± 1.2 (CFU/g of fresh root) with P. putida L2 and 3.9 × 10 3 ± 1.6 and 3.8 (CFU/g of fresh root) with S. pavanii L8, respectively.

Discussion
Biochar amendments have been reported to elicit complex responses in plants [4,6,15], including plant growth stimulation or suppression of several soil-borne diseases [47,48]. However, the beneficial effect depends on the type of biochar feedstock, the technique of preparation (e.g., pyrolysis or carbonization), and finally, concentration [15,16]. Overall, our experiment showed that the shoot and root biomass of lupin responded differently to types of biochar and biochar concentrations, i.e., MBC char produced more benefits to lupin growth as compared to WBC char. Reibe et al. [33] reported similar observation for MBC char amendments increasing dry biomass of wheat as compared to WBC char or plants grown without any biochar application. Joseph et al. [49] reported that the interaction of biochar with environmental conditions is another crucial requirement for revealing contrasting effects, which might depend on the physicochemical properties of biochars. For example, Butnan et al. [25] observed reduced plant growth on a sandy Ultisol amended with eucalyptus wood-derived biochar produced by pyrolysis (800 • C), whereas biochar produced at a lower temperature (350 • C) provided higher benefits. Shen et al. [50] reported that the addition of biochar from willow woodchips increased plant growth of Lotus pedunculatus cv barsille, whereas pine-based biochar did not show any positive effect on plant growth.
There are also numerous reports on the positive effect of biochar in reducing plant pathogens [51], e.g., R. solani of cucumber [16] or F. oxysporum f. sp. lycopersici of tomato [14]. However, some biochar types showed an increased disease incidence by providing favorable conditions for pathogenic fungi, e.g., 3% green waste biochar increased the disease incidence of tomato wilt caused by F. oxysporum f. sp. lycopersici [14]. Correspondingly, we observed an increased disease incidence of lupin wilt caused by F. solani under WBC char, whereas no diseased plants were observed in control plots. However, the mechanisms of biochar to suppress or induce soil-borne diseases were not sufficiently revealed because they depend upon several factors, including chemical and physical characteristics of biochar [16] as well as soil conditions and properties. In earlier studies, Sanogo and Yang [52] reported that the development and growth of Fusarium species was faster at higher soil pH 8.2 compared to that at lower soil pH 5.7.
The capability of endophytic bacteria living within plant tissues to inhibit plant pathogens was reported in numerous studies [22,53,54]. Silva et al. [55] observed a biological control of coffee leaf rust caused by Hemileia vastatrix with endophytic bacteria such as Bacillus megaterium and Microbacterium testaceum. In another study, Bacillus cereus decreased wilt symptoms of tomato caused by F. oxysporum [56]. In our study, endophytic bacteria P. putida L2 and S. pavanii L8 were able to reduce lupin root rot caused by F. solani in WMB and MBC amended soil. In a previous study, the endophytic Bacillus subtilis NUU4 with antagonistic activity against F. oxysporum, F. solani, and F. culmorum reduced disease incidence of chickpea root rot [54]. The plant inoculation with P. putida L2 and S. pavanii L8 increased lupin root and shoot biomass grown in soil without biochar. The root biomass of lupin was significantly increased by P. putida L2 under both MBC and WBC char. Lupin treated with Bradyrhizobium sp. and growing in soil amended with biochar had higher root and shoot biomass as compared to un-inoculated control plants or direct inoculation with Bradyrhizobium sp., alone [9]. Correspondingly, soil amendment with biochar from citrus wood provided favorable conditions for bacterial proliferation under sweet pepper (Capsicum annuum L.) [18]. Pietikäinen et al. [57] reported that the survival of bacteria, which were adsorbed to biochar surfaces, was due to biochar properties protecting bacteria in the soil. In another study, the number of rhizobial cells in soil under soybean were increased by the addition of biochar mixed with compost [58]. Hale et al. [59] observed that pinewood biochar based inoculum of E. cloacae UW5 resulted in a significant increase in cucumber root branching and total root length giving evidence for the role of biochar in effects on root architecture. The ability of endophytic bacteria colonizing the internal part of plants to stimulate the root system by better nutrient uptake was reported in numerous studies [53,54,60,61]. The colonization of inoculated bacterial isolates in the root system is an important trait, especially in a highly competitive environment [62,63]. Moreover, it is the very first step in beneficial interactions of the plant with a microbe. The inoculated endophytic bacteria were able to survive in the rhizosphere of lupin grown in soil without biochar and biochar amendment for 30 days. It has been previously reported that improved plant-microbe interactions resulted from biochar effects on soil microbial diversity and activity [64], e.g., biochar maintains bacterial proliferation residing inside pores by supplying nutrients and improving aeration [65].
Several mechanisms by which endophytic bacteria enhance plant growth, improve stress tolerance, and protect plants from various diseases were described. These include the production of phytohormones such as indole 3-acetic acid [9,54], antifungal metabolites [62,66], and cell wall degrading enzymes such as cellulase or chitinase [67]. In our previous work, the endophytic strain B. subtilis NUU4 that reduced root rot incidence in chickpea and stimulated plant growth demonstrated an antagonistic activity against F. oxysporum, F. solani, and F. culmorum and produced IAA, as well as cell wall degrading enzymes. Similar observations were found by Abdallah et al. [56], where the wilt symptoms of tomato caused by F. oxysporum were decreased by B. cereus antagonistic against F. oxysporum f. sp. lycopersici. The endophytic bacteria that synthesized IAA stimulated the root system architecture and also increased root hairs, which resulted in better absorption of nutrients [68].

Conclusions
The results presented in this study showed that soil amended with 2% MBC and HTC improved root and shoot growth of lupin. However, the disease symptoms of lupin root rot occurred in soil amended with WBC char at a rate of 2% and 3%. Plants under HTC char were healthy, and no disease incidence was found. The endophytic bacteria P. putida L2 and S. pavanii L8 isolated from lupin were able to reduce root rot caused by F. solani under WBC amended soil. Furthermore, the lupin growth was also increased by inoculation with endophytic bacteria in MBC and WBC amended soil treatments. The soil amended with MBC and WBC supported better survival of inoculated bacteria in the rhizosphere of lupin. Future research is required to reveal the mechanisms of biochar-induced disease incidence in more detail, as well as the traits endophytic bacteria use to control lupin root rot caused by F. solani in soil amended with biochar, especially considering soil physicochemical properties.