Biochar exudates do not present negative effects on the survival and infectivity of nematodes

To evaluate the nematicidal effect of biochar on M. graminicola, the nematodes were incubated in different concentrations of biochar exudates. Significant differences in nematode mortality were not observed between biochar exudates (6.6 ± 0.7 %) and water (7.2 ± 0.6 %) 24 h after initiation of the bioassay at doses ranging from 0.3 to 5 % biochar (Fig. 1a). Similar results were also observed when the nematodes were incubated in biochar exudates for 72 h (Fig. 1a). These data suggest that biochar exudates do not have a direct nematicidal effect on M. graminicola at the doses tested.

Fig. 1 Direct effect of the biochar exudates on the behavior of M. graminicola (Mg). a Percentage of dead juveniles 24 h and 72 h after incubation in various concentrations of biochar exudates and water. b Penetration and development of biochar-incubated and water-incubated M. graminicola in rice roots. c Biochar-incubated and water-incubated nematodes were inoculated on rice roots and photographed at 7 dpi. d Biochar-incubated and water-incubated nematodes were inoculated on rice roots and photographed at 14 dpi. The bars in the different graphs represent the mean ± SE of the data from three independent biological replicates, each containing 6 individual plants. Different letters indicate statistically significant differences (Duncan’s multiple range test at p ≤ 0.05). J2: second stage juveniles. J3: third stage juveniles. J4: fourth stage juveniles Full size image

To verify whether biochar can hamper the infectivity of RKNs, the nematodes treated with biochar exudates or water (as control) were inoculated in rice roots. The nematode penetration and development were recorded at 7 and 14 dpi (Fig. 1b). At 7 dpi, most of the nematodes developed to third-stage juveniles (J3). The mean number of nematodes inside the roots and their development was not different between the biochar-exudate treated and control nematodes (Fig. 1b and c). At 14 dpi, most of the nematodes had developed into adult females. Again, significant differences were not observed in the number of adult females or the total number of nematodes in the biochar exudate-treated and water-treated nematodes (Fig. 1b and d). The ratio of adult females among the biochar exudate-treated nematodes (91.6 ± 7.4 %) was similar to that in the water-treated nematodes (93.2 ± 7.1 %). Overall, our data show that incubation of M. graminicola in biochar exudates did not inhibit their penetration or delay their development inside the rice roots.

Soil amended with biochar reduces the infection of M. graminicola in rice without restraining plant growth

Recently, relatively low concentrations (1 %) of four biochars prepared from two feedstocks at different pyrolysis temperatures were found to suppress the damping-off of Rhizoctonia solani in beans, whereas a higher concentration (3 %) provided ineffective disease protection [30]. Thus, the effect of different biochar doses deserves more attention. To evaluate the potential of biochar as a priming agent, different concentrations of biochar were added to the SAP-substrate, and plant susceptibility was evaluated at 14 dpi. The results revealed that the biochar amendment reduced the number of galls per g of root and the number of nematodes per g of root at all of the tested concentrations (Fig. 2a). However, the best effect was observed at a concentration of 1.2 % biochar in SAP. Therefore, all further experiments were executed with this optimal biochar concentration of 1.2 %.

Fig. 2 Effects of biochar amendments on plant growth and nematode infectivity in rice roots. a Root galls and nematodes per gram root in biochar-amended (different concentrations) and non-amended rice roots were counted at 14 dpi. b Plant height and fresh weight were measured at 14 dpi. c Root galls per plant on the 1.2 % biochar-amended and non-amended rice roots were counted at 14 dpi. d Nematodes per root in different developmental stages in the 1.2 % biochar-amended and non-amended rice roots were counted at 14 dpi. The bars in the different graphs represent the mean ± SE of the data from three independent biological replicates, each containing 6 individual plants. Different letters indicate statistically significant differences (Duncan’s multiple range test at p ≤ 0.05) Full size image

In subsequent experiments, the growth parameters were assessed by analyzing the length and fresh weight of the roots and shoots of 4-week-old plants (Fig. 2b). When comparing the RKN-infected plants with non-infected plants, slight but significant reductions in the root length and total plant height were observed in the RKN-infected plants. Although 1.2 % biochar alone did not have a significant effect on the analyzed growth parameters, the biochar amendment partially alleviated the negative effects caused by the RKN-infection.

At the optimal concentration of 1.2 %, amendments of biochar significantly reduced the total number of root galls at 14 dpi (Fig. 2c). In addition, the development of nematodes in biochar-amended roots was slightly delayed. The number of adult females in biochar-amended roots was slightly lower than that of non-amended plants, whereas a higher number of fourth-stage juveniles (J4s) were observed in biochar-amended roots compared with that of non-amended plants (Fig. 2d).

Root exudates often attract nematodes and trigger egg hatching in certain plant-parasitic nematode species [31]. To verify whether biochar impedes the ability of the plant to attract M. graminicola, rice roots were drenched with biochar exudates or water 1 d before inoculation. At 9 hpi, approximately 20.2 ± 3.1 % of the nematodes were attracted to the biochar-treated root tips, which was not significantly different from those attracted to the non-amended root tips (23.3 ± 3.2 %) (p>0.05) (Fig. 3a, b). This result indicates that the tested biochar exudates do not prevent the attraction of M. graminicola to rice.

Fig. 3 Effect of biochar on the attractiveness of rice roots to M. graminicola and microscopic observations of giant cells induced in the root system. a Attraction of M. graminicola towards the root tips of rice after root drenching with 1.2 % biochar exudates or water were observed under a Leica stereomicroscope with a DFC400 camera. b Nematodes in the vicinity of the root elongation zone were counted at 9 hpi. The bars represent the mean ± SE of the data from 6 replicates. No significant differences were found (Duncan’s multiple range test at p > 0.05). c Sections of giant cells in the biochar-amended root galls and non-amended root galls were stained with toluidine blue and observed at 7 dpi under an Olympus BX 51 microscope with a ColorView III camera. Multiple sections of 10 galls were evaluated and the figure shows one representative section for each treatment Full size image

A microscopic analysis of the nematode feeding sites inside the galls revealed that significant morphological differences did not occur in the giant cells formed in the biochar-amended roots versus the non-amended roots. Most of the giant cells were still enlarged cells with multiple nuclei, dense cytoplasm, and thickened cell walls (Fig. 3c).

These data demonstrate that biochar amendments at a concentration of 1.2 % delay the development of the RKNs but do not change the root attractiveness or the giant cell morphology. However, at this concentration, biochar amendments to the soil can reduce the negative effect of RKNs on plant growth.

Biochar amendment does not induce callose deposition in root galls

The addition of BABA to protect rice plants from RKNs was previously shown to be correlated with enhanced glucan synthase-like gene (OsGSL1) mRNA levels and callose deposition in the gall tissue [14]. To investigate whether biochar has a similar mode of action, the expression of this callose synthase-encoding gene, OsGSL1, was investigated by quantitative reverse transcriptase PCR (qRT-PCR) in biochar-amended and non-amended plants. At 24 hpi, the transcription level of OsGSL1 was significantly down-regulated in the biochar-amended plants compared with the control plants (Fig. 4a). Significant differences were not observed in inoculated plants, whether biochar-amended or non-amended, although in both cases, a trend towards lower expression of this gene was observed. Confirming these results, the prominence and density of callose spots in biochar-amended galls were similar to those in the non-amended galls at 7 dpi (Fig. 4b and c). These data suggest that biochar amendments do not induce callose deposition after nematode invasion.

Fig. 4 Effect of biochar-amendment to the growth medium on callose biosynthesis in the rice root system. a The relative transcript levels of a callose biosynthesis gene (glucan synthase-like gene, OsGSL1) at 24 hpi were analyzed using qRT-PCR. The gene expression levels were normalized using three internal reference genes, OsEXP, OsEif5C and OsEXPnarsai. The data are shown as the relative transcript levels normalized to the control roots (expression level in the control set at 1). The bars represent the mean expression level ± SE from two independent biological replicates , each containing a pool of 6 plants. Asterisks indicate significantly different expression levels in comparison with the control roots. b Callose deposition in the root galls at 7 dpi was examined under UV light using a Nikon Eclipse Ti-E epifluorescence microscope (excitation 390 nm; emission 460 nm). c Quantification of callose deposition was performed using ImageJ software. The data presented are the mean ± SE of two independent experiments, each performed using ten galls. Different letters indicate significant differences (Duncan’s multiple range test at p ≤ 0.05) Full size image

Biochar amendment induces H 2 O 2 accumulation but not lignification in root tissues

H 2 O 2 is an important reactive oxygen species (ROS) and essential to the induction of defense responses in plants. This experiment was conducted to investigate whether biochar is capable of generating ROS for the induction of defense against M. graminicola. First, the H 2 O 2 levels were measured in the plant roots at three different time points, and the results showed that biochar amendments alone led to higher H 2 O 2 levels in the rice roots (Fig. 5a). Upon Mg-inoculation (in non-amended SAP), an increase in the H 2 O 2 levels was also observed. However, in biochar-amended inoculated plants, the H 2 O 2 levels increased to significantly higher levels at all of the investigated time points, indicating a priming effect. A quantitative analysis of OsRbohB, an NADPH oxidase gene involved in the plant immune response [32], showed that the transcription level of OsRbohB was significantly up-regulated in plants that received biochar amendment alone compared with non-amended non-inoculated control plants at 24 hpi (Fig. 5b). However, significant differences were not observed in biochar-amended inoculated plants and non-amended inoculated plants. Most likely, the root knot nematode interferes with the induction of this gene or its induction happens at other time points than those studied here.

Fig. 5 Effect of biochar-amendment to the growth medium on H 2 0 2 accumulation and lignin levels in the rice roots. a H 2 O 2 content per gram of root was measured upon reaction with KI and detection using a CLARIOstar Microplate Reader at 390 nm. The bars represent the mean ± SE of four replicates, each containing a pool of six roots. Different letters indicate significant differences (Duncan’s Multiple Range Test at p ≤ 0.05). b Relative transcript levels of the H 2 O 2 synthesis gene (OsRbohB) at 24 hpi were analyzed using qRT-PCR. The gene expression levels were normalized using three internal reference genes, OsEXP, OsEif5C and OsEXPnarsai. The data shown are the relative transcript levels compared with the control roots (expression level set at 1). The bars represent the mean expression level ± SE from two independent biological replicates, each containing a pool of 6 plants. Asterisks indicate significantly different expression levels in comparison with water-treated control roots. (p ≤ 0.05). c Lignin content in the roots of rice amended with 1.2 % biochar or water was determined using the acetylbromide assay. Root samples were collected before inoculation (0 h) and 24 hpi. The bars represent the mean ± SE of the lignin content of 6 plants. Different letters indicate significant differences (Duncan’s multiple range test at p ≤ 0.05) Full size image

The increased production of H 2 O 2 is known to cause the polymerization of monolignols by peroxidase and subsequent formation of lignin [33]. Lignin confers mechanical strength to plant secondary cell walls, which contributes to basal defenses against plant-parasitic nematodes [34]. Prior to nematode inoculation (0 h), the lignin level in the roots receiving the biochar amendment alone was similar to that in the non-amended roots (Fig. 5c). At 24 hpi, slightly stronger lignification was observed in the biochar-amended inoculated roots, although the difference was not statistically significant. These data indicate that biochar amendments do not strongly promote lignin synthesis.

Biochar-induced defense in rice against M. graminicola is partly mediated by the activation of ET signaling

ET can be produced from the pyrolysis of biomass, although the production of ET varied drastically across different evaluated biochars [35]. To investigate the importance of the ET pathway in biochar-induced resistance against RKNs, the expression levels of genes involved in ET responses (OsERF70, OsERF1, OsEBP89), ET biosynthesis (OsACS1, OsACO7) and ET signaling (OsEIN2) were analyzed.

The transcription of the ET response genes OsERF1 and OsEBP89 was significantly up-regulated in the biochar-amended plants (Fig. 6a), whereas OsERF70 was not significantly affected by these treatments. The two ET-biosynthesis genes showed inconsistent results, with OsACO7 slightly induced by all treatments and OsACS1 repressed by the treatments, although none of these values were significantly different from the control plants (Fig. 6b). Transcription of the ET signaling gene OsEIN2 showed a minor but non-significant induction following all treatments (Fig. 6c).

Fig. 6 The effect of biochar-amendment in the rice growth medium on the ET-pathway in the rice roots. a Relative expression levels of OsERF1, OsEBP89, OsERF70, which are involved in the ethylene response pathway, were analyzed at 24 hpi using qRT-PCR. b Relative expression levels of OsACO7 and OsACS1, which are involved in the ethylene biosynthesis pathway, were analyzed at 24 hpi using qRT-PCR. c Relative expression levels of OsEIN2, which is involved in the ethylene signaling pathway, were analyzed at 24 hpi using qRT-PCR. The gene expression levels were normalized using three internal reference genes, OsEXP, OsEif5C and OsEXPnarsai. The data shown are the relative transcript levels compared with the control roots (expression level set at 1). The bars represent the mean expression level ± SE from two independent biological replicates and three technical replicates, each containing a pool of 6 plants. Asterisks indicate significantly different expression levels (p ≤ 0.05). 1.2 % Biochar + Mg, 1.2 % biochar amendment plus M. graminicola inoculation; 1.2 % Biochar, 1.2 % biochar amendment alone; Mg, M. graminicola inoculation alone; control, non-amended and non-inoculated. d Effect of an Ein2b-RNAi mutant, which is deficient in ethylene signaling, and the wild type Nipponbare on nematode infection at 14 dpi. The bars represent the mean of the data from three independent biological replicates, each containing 6 plants. Different letters indicate significant differences (Duncan’s multiple range test at p ≤ 0.05) Full size image

To obtain a more detailed understanding of the role of the ET response in biochar-induced defenses against RKNs, an Ein2b-RNAi line deficient in ET signaling was investigated (Fig. 6d). Confirming our earlier observations (Fig. 2a), the number of nematodes at 14 dpi was reduced in the biochar-amended Nipponbare plants. However, significant differences were not observed between the biochar-amended and non-amended plants in the Ein2b-RNAi line. These results imply that the ET signaling pathway is required for biochar-induced defense against M. graminicola in rice.