Introduction

Tomato (Lycopersicon esculentum Mill.) is a short duration, high yielding crop as compare to others and due to its high profit, it has become more popular and the area under its cultivation is increasing day by day (Naika et al. 2005). Tomato crop is attacked by a large number of plant pathogens; however, plant parasitic nematodes are major pests of tomato crop worldwide.

Plant-parasitic nematodes are the obligate, microscopic, bio-trophic organisms causing destructive yield loss in vast range of crops such as potato, chickpea, tomato, wheat, sugar beet etc. Worldwide the total annual yields losses due to plant-parasitic nematodes occur in various crops have been estimated over $157 billion (Abad et al. 2008). Plant-parasitic nematodes can cause yield losses up to 20% and they cast disturbing effect on farmer in developing countries (Atkinson et al. 1995). RKNs feed inside the root system and form galls which consist of numerous giant cells on the vasculature of the plant roots. These giant cells are the only source of nutrition for the sedentary stages of RKNs (Jones and Payne 1978; Ali et al. 2015). Among a huge number of RKNs species, M. incognita is the most dangerous specie of nematodes which can parasitize almost all the cultivated crop plants including tomato (Abad et al. 2008).

M. incognita results in significant losses in both quality and quantitiy of the tomato produce and some potential nematode strategy must be developed to minimize these losses. Various strategies have been employed for controlling parasitic nematodes in plants (Ali et al. 2017). Synthetic chemicals have been widely used in controlling parasitic nematodes in the field which have ultimate health and environment hazards. Soil amendments with different organic and inorganic material have been largely used. Recently several scientists studied the application of biochar for controlling various pathogens in different crop plants.

Biochar (BC) is a charcoal like material which has excessive amount of carbons and its derivatives. It is acquired by the burning of organic biomass at very high temperature in a special type of furnace. It can be helpful for the sequestration of carbon by adding huge quantity of carbon or burned organic compounds into the soils which are hard to decompose (El-Naggar et al. 2019). Biochar remains in the soil for a very long duration and can capture atmospheric carbon for a prolonged period of time (Elad et al. 2010; Vaccari et al. 2011). It is reported that biochars obtained from different feedstocks led to increase in plant biomass and economic yields in different crop plants (Hussain et al. 2017). For instance, Kammann et al. (2012) observed a significant increase in biomass in biochar amended soils as compared to controls in perennial ryegrass (Lolium perenne L.). Similarly, Rondon et al. (2007) displayed that addition of biochar to a tropical, low-fertility soil led to 22% enhanced nitrogen fixation in bean plants (Phaseolus vulgaris) in addition to significantly improved biomass production and bean yield. Graber et al. (2010) reported that improved plant growth and fruit yield in pepper and tomato due to biochar amendment may shift the microbial community structure towards beneficial plant growth promoting rhizobacteria (PGPR) and plant growth promoting fungi (PGPF) in pepper.

The literature displays the promising effects of biochar to induce systemic resistance against many plant pathogens (Elad et al. 2010; Vaccari et al. 2011; Zwart and Kim 2012). For instance, biochar application in the soil led induced resistance in two tree species (Quercus rubra and Acer rubrum) against stem lesions caused by Phytophthora spp. (Zwart and Kim 2012). Likewise, soil amendment with biochar reduced the susceptibility of pepper and tomato plants against some foliar fungal pathogens like Leveillula taurica and Botrytis cinerea in addition to reduced attack of broad mites in pepper plant (Elad et al. 2010). Moreover, application of biochar in potting material under different conditions led to enhanced resistance in rice against root knot nematode, M. graminicola (Huang et al. 2015). By keeping these reports in mind, the present study was designed to control RKN and improve plant growth and development through the application of biochar in tomato. For this purpose, the potential of biochar from different feedstocks was assessed against RKN M. incognita in tomato under in vitro and pot experiments.

Materials and Methods

Pyrolysis of feedstocks for biochar production

Three feed stocks i.e., sugarcane bagasse, rice husk and wheat straw were used for the production of biochar. Wheat straw was collected from the agricultural farm of department of plant Pathology, University of Agriculture, Faisalabad while rice husk and sugarcane bagasse were taken from rice dehusking and sugar mills, respectively. These feed stocks were sun dried to retain moisture up to 15% before biochar preparation.

The feedstocks were pyrolyzed at temperature 450°C with retention time of 30 min after a gradual rise of 10°C min⁻¹ for production of biochars in a laboratory setup Muffle furnace (Gallonhop, England) following the protocol described by Sanchez et al. (2009). The biochar was ground and sieved before further experimentation and analysis.

Scanning electron microscopy (SEM) & X-rays diffraction (XRD)

The structural and surface analysis of the particles of three biochars was visualized by mean of SEM. For this purpose, the samples were observed under 30KV Scanning Electron Microscope (JSM5910, JEOL, Japan) at 500,1000,5000 and 10,000x. SEM was performed to examine the structural properties of the samples. The XRD pattern was developed through XRD spectroscopy using a X-ray Diffractometer (Philips PANalytical X’Pert Powder) with CuKα source radiation (λ = 1.5405 Å) over the angular range 20° ≤ 2θ ≤ 80°, operating at 30 kV and 10 mA. The scanning peaks were collected in the range of 20~80°, then the analysis of the data was done through MDI Jade 6.0.

Extraction and identification of nematodes

Infected roots of tomato were collected from the field area of Department of Plant Pathology. These infected roots were washed to clean the debris. The roots were cut into small pieces and put into a jar. 10% solution of NaOCl was prepared and added into the jar and jar was shaken well for 4 min. Three sieves were arranged in order from top to bottom (150, 250, 400 mesh. After that jar solution including roots was poured on the upper sieve and washed enough to remove NaOCl. Eggs were collected from the last (400 mesh) sieve and placed them on filter paper which touched the water surface of a tray. The filter paper was covered and incubated for 48 hours at 23°C. Freshly hatched Meloidogyne incognita juveniles were identified under automated upright microscope (LEICA DM5500 B) using software name (LAS V4.12). 2^nd stage juveniles (J₂) were identified on the basis of body length (346–463 µm), tail length (40–60 µm), (Dorsal esophageal gland orifices) DGO to stylet base (2–3 µm) and tail shape by Karssen (1996). Larvae counting was done using stereoscope.

In-vitro experiment

Suspension of 1 mL freshly hatched juvenile (200 J2) was incubated with 1 mL of biochar exudate taken from various concentration of biochar (0.3, 0.5, 1.2, 2.3 and 3% from each of the 3 feed stocks i.e., wheat-straw, rice husk and sugarcane bagasse) in 3.5 cm span wells on a 6 well culture plates. These exudates and their concentrations were prepared and used according the method reported by Huang et al. (2015). After 24 h, 48 h and 72 h, 1 N NaOH was added into the suspensions, and nematode that retorted to NaOH by change their bodies shapes within 3 min were consider alive, however nematodes which failed to move after adding NaOH were consider as dead according to Chen and Dickson (2000). This experiment was performed in 6 replications for each feedstock to acquire reliable results.

Growing and transfer of tomato nursery

Tomato cultivar i.e., Money Maker, was grown in the pots in the sandy loam soil with the percentage of sand =75%, silt=23% and clay=7%. Three biochars with three concentrations 2, 3 and 5%, respectively were used for the amendment into the potting soil. Each concentration was mixed in 20 g, 30 g and 50 g w/w in the pot capacity of 1000g soil to make concentrations. Before this, soil was properly sterilized with formalin @ 1:5 for a week to minimize the soil nematodes population. After 3 weeks of sowing, tomato seedlings were transplanted in earthen pots (1 kg capacity) with 3 treatments, 3 concentrations, 4 replications and leaving two controls of each which had neither biochar no-inoculum as negative control and the other was +ve control which had only nematode inoculum in completely randomized design fashion (CRD). Plants were irrigated regularly along with appropriate fertilization.

Nematode inoculation

Two-week-old tomato nursery was inoculated with newly hatched juveniles (J2) of M. incognita at the rate of 1000 larvae per plant. Nematodes were inoculated in pot by making three holes with a glass rod around the stem of the plant in soil near root zone without damaging root system.

Effect of various biochar treatments on plant growth parameters under nematode infection

Plant growth parameters like plant height (cm), fresh shoot weight (g), dry shoot weight (g) were recorded after 6 weeks of inoculation. Similarly, below ground parameters like root length (cm), root fresh and dry weights (g) were measured. Additionally, disease parameters were also recorded at the termination of experiment after 8 weeks of inoculation. To measure dry weights, samples were dried at 80°C in dry oven for 48 h.

Assessment of nematode development on plant roots in various treatments

The data regarding various parameters like galling index, egg mass index, number of females were evaluated after 8 weeks post inoculation in different treatments of biochar application. Plants were uprooted and taken to the lab and the roots rinsed with tap water to remove debris. Galls were evaluated by the scale of 0–10 described by Bridge and Page (1980) Egg masses were stained using phloxine B (Holbrook et al. 1983) and quantified using a scale of 1–9 where: 1 = no egg masses, 2 = 1–5, 3 = 6–10, 4 = 11–20, 5 = 21–30, 6 = 31–50, 7 = 51–70, 8 = 71–100, and 9 = >100 egg masses per root system (Sharma et al. 1994). Number of females was counted visually by using stereomicroscope in a root system.

Statistical analysis

The collected data were subjected to factorial analysis of variance (ANOVA) under completely randomized design (CRD) using Statistix 8.1. The means of various treatments were compared using least significant difference (LSD) test at 95% level of confidence (P ≤ 0.05). Moreover, percentage decrease was calculated from control to assess the efficacy of various treatments for nematode control in tomato.

Fig. 1: Images of different biochars under scanned electron microscope (A) WS at the magnification of 500, 1000, 5000 and 10,000x (from left to right) (B, C) RH and SCB with the same magnification respectively

Fig. 2: X-rays diffraction analysis of three biochar to check the structural nature of biochars. Where A: XRD of WS, B: XRD of RH and C: XRD of SCB

Results

SEM and XRD of biochars from different feedstocks

The formation of a bundle shaped tube on RH biochar created uneven sized pores having a diameter range between 50 μm when visualized at four different magnifications, pores were well distributed they may have capacity to provide habitat to a number of beneficial microorganisms shown in (Fig. 1b). On the other hand, the pores of SCB were seemed rough at 500x while under 1000x and above, the pores looked like in an arranged manner (Fig. 1c). Compared with biochar from WS under same magnifications appeared tiny carbon slices or ash particles were clearly adhered with each other in scattered form (Fig. 1a).

X-rays diffraction (XRD) is a procedure by which we can analyze the structure of a substance and the abundance of elements present in it. The X-Ray diffraction patterns of various biochars from different sources are displayed in Fig. 2. It is obvious that the only high peak in the RH derived biochar at 2θ=72.3° shows the presence of high amorphous silica present in it while the other peaks show no substantial height. It means the substance which was made on temperature 450° is sub-crystalline in nature shown in (Fig. 2B). However, in case of SCB biochar XRD analysis, broad peaks were located between 23.3–40.4° which indicates the amorphous nature of a substance and some inorganic compounds like (C-200, K₂SO₄, Ca (OH)₂, MnO₂ and SiO₂) where the highest peak was again silica diffraction observed at 72.5° has shown in (Fig. 2C). On the other hand, WS showed more crystalline nature than the other two biochars. The small sharp peaks are clearly obvious between the 27.9 –68.3° and mostly represent the inorganic compounds which exist in oxide form (SiO₂, MnO₂, Al₂O₃) and some other compounds like (K₂SO₄, Ca (OH)₂, and KCl) are also present in wheat straw biochar. The highest peak was observed at 26.5° for SiO₂ (Fig. 2A).

Nematicidal potential of different biochars through in vitro experiment

Fig. 3: Percentage of dead nematodes showed in different concentrations (0.3, 0.5, 1.2, 2.3 and 3% of WS, RH and SCB along with control (nematodes incubated in only water) (A) After 24 h (B) After 48 hours (C) After 72 h

Fig. 4: Regression analysis between No. of females and No. of galls with (A, B) Fresh shoot weight (g); (C, D) Fresh root weight (g); (E, F) Shoot length (cm); (G, H) Root length (cm) during the treatments of three biochars

To assess the toxic effects of biochars on M. incognita, freshly hatched 2^nd stage juveniles (J2) were incubated with the different concentrations (0.3, 0.5, 1.2, 2.3 and 3%) of exudates from three different biochar and nematodes incubated in distilled water were kept as control (Fig. 3). After 24, 48 and 72 h, significant nematode mortality was found in WS, RH and SCB exudates of above-mentioned concentrations. Very minute difference in mortality was observed in treated and control plates. After 24 h maximum dead juveniles were evaluated in (1.2% SCB, control and 3% SCB) respectively shown in (Fig. 4A). Similarly, the maximum death count after 48 h was observed in same plates mentioned earlier (Fig. 4B). While after 72 h maximum mortality of nematodes were observed in 1.2% SCB and control plates shown in (Fig. 4C) So, the data revealed that biochars have somehow statistically significant effects on nematode mortality but not efficiently reduced as compare to control.

Table 1: Effect of biochar concentrations on RKN population and plant biomass parameters

Values are mean of four replicated plots. Means followed by different letter(s) within a column are significantly different using LSD at P = 0.05

*SL= Shoot length, *RL= Root length, *FSW= Fresh shoot weight, *DSW= Dry shoot weight, *FRW= Fresh root weight, *DRW= Dry root weight

Table 2: Effect of different biochar’s concentrations on RKN population

Values are mean of four replicated plots. Means followed by different letter(s) within a column are significantly different using LSD at P = 0.05 *EMI= Egg mass index, *GI= Galling index

Reduction % = [(Cf (control) - Cf (treatment)/Cf (+ control)] × 100

Incorporation of biochar in potting material leads to enhanced plant growth

The data regarding the effect of different concentration of various biochars on plant growth parameters are given in Table 1. The results demonstrated that shoot length was significantly (P < 0.05) increased in all of the RH treatments including 3, 5 and 2% concentrations of this biochar. While, the lowest value of shoot length was recorded in positive control (16.6 cm) where only nematodes were applied. For the FSW and DSW the 3% of RH found best with maximum mean values of 56.7 g and 22.4 g respectively. However, the least values in FSW and DSW after positive control were found in 3% of WS and 2% SCB respectively. Similarly, for the root system parameters like RL, FRW and DRW demonstrated significant results at 95% level of confidence for different treatments of biochars. Significantly highest values of RL, FRW and DRW (27.3 cm, 30.1 and 9.3 g respectively) were observed in 5% of SCB. Likewise, 3% RH showed the increasing value in RL, FRW and DRW after 5% SCB with mean values (25.0, 28.7 and 7.9 g). However, the FRW and DRW in positive control demonstrated minimum values (19.8 and 5.5) respectively due to root abbreviation and heavy galling on the roots. Moreover, the lowest values after positive control were found in 2% WS with 15.0 cm, 10.8 g and 2.7 g beside this in negative control plants having no biochar and no inoculum showed overlapping values with some treatments of biochars (Table 1).

Addition of biochars led to enhanced resistance against M. incognita in tomato

Various nematode reproduction and establishment attributes were studied in response to different concentrations of biochars (Table 2). Number of galls, number of egg masses per root system, galling index, egg mass index and number of females were significantly (P ˂ 0.05) different in all the treatments. The lowest number of females, galls, galling index, egg masses and egg mass index were found in 3% RH treatment. Minimum number of females (52.3) was examined in 3% RH while the highest number of egg masses were recorded in 5% WS which were (120.6) while the nematode control treatment showed maximum value for egg masses (170.6). In addition, highest number of females among all the biochar treatments were recorded in 3% WS with mean value (117.6). Likewise, maximum number of galls in treated plants was recorded in 2% SCB. Beside this the highest number of nematode establishment based on number of females, egg masses and number of galls in root system with values (138.3, 170.6 and 120) respectively was examined in positive control; however, the negative control displayed no infection of nematodes due to absence of nematode inoculum. Percentage of decrease of nematode parameters was determined and number of females, egg masses and galls were 62.1, 77.5 and 83.1% decreased, respectively as compared to positive in 3% RH treatment. Response of various growth parameters was correlated with different nematode development parameters. Fresh shoot weight, fresh root weight, shoot length and root length showed a decreasing trend in response to increasing number of females and number of galls in tomato (Fig. 4). Although, fresh root weight showed little effect, it increased in females and galls (Fig. 4C–D).

Discussion

Surface and structural assessment of various biochars was done using SEM and XRD techniques. SEM and XRD analyses demonstrated that the wheat biochar displayed sub-crystalline nature and exhibited more inorganic compounds as compare to other two biochars. While in rice husk biochar the silica was present in high concentration which might be responsible for improving plant biomass when it absorbs into the soil which highly favors the findings in the present study. This could be due the reason that formation of phytolith in plants is highly dependent on availability of silica (Nawaz et al. 2019). Moreover, the silica compounds would not loss after pyrolysis process (Houben et al. 2013). Our findings showed that other miscellaneous inorganic compounds were absent in rice husk biochar which were present in other biochars. Sub-crystalline nature of WS biochar developed at high pyrolysis temperature displayed high peaks for silica and K₂SO₄ which is in accordance with the results of Yuan et al. (2011). Most of the compounds present in SCB and WS biochar were in oxide form. These results were comparable with some recent findings based on XRD analysis of different biochars regarding the possible abundance of different compounds at different theta angles (Trubetskaya et al. 2016: Mohan et al. 2018).

Plant pathogenic nematodes are notorious pests of economically important crop plants (Ali et al. 2015). Various management practices have been used to control the nematode population below the damaging levels including cultural, chemical, biological and transgenic approaches (Ali et al. 2017). To investigate the potential of biochar as a significant nematode control strategy, we conducted a series of experiments including in vitro nematicidal assay and pot experiments. To check the nematicidal effect of different biochars, the exudates of biochar were obtained and assessed for in vitro nematicidal effects on J2s of M. incognita. The result of this experiment concluded that there are no such chemicals present in the tested biochar, which can have direct effects on nematode mortality. Although, very minute difference in nematode mortality has been found between treated and control treatments. Similar experiment was designed by the Huang et al. (2015) who also reported no direct effect of biochar exudates on M. graminicola at different concentrations. Moreover, very recently in vitro treatments of biochar did not show any direct toxicity or suppression in mycelia of two fungi pathogenic to tomato plant (Jaiswal et al. 2018).

Real time efficacy of various biochars with different concentrations was investigated through a pot experiment based on soil application of the biochars. Soil amendments have largely been used for the purpose of pest and disease control in different crop plants. Amendment of soil with biochar led to enhanced plant growth due to sequestration of carbon into soil which not only improves soil fertility but also plays vital role in suppressing the soil borne pathogen populations (Lehmann and Joseph 2009; Zimmerman 2010). Our results demonstrated that biochar amendment led to significant improvement in the plant growth and development. Previously, application of biochar has increased plant growth in various plant species like pine and alder (Robertson et al. 2012), peanut (Agegnehu et al. 2015), tomato (Vaccari et al. 2015), wheat (Akhtar et al. 2015) and soybean (Sanvong and Nathewet 2014). However, contrastingly, some studies which demonstrated no significant effect of biochar on plant growth (Chan et al. 2007; Zwieten et al. 2010). Another study revealed that the biochar amendment in the soil can enhance the grapevines biomass in addition to reduction in nematode population by accumulating beneficial microbes in the soil (Rahman et al. 2014). A study conducted by Abrishamkesh et al. (2015) found that amendment of RH biochar can increase the above-ground and below-ground biomass of lentil plants which supports our results where RH biochar showed significant results in promoting overall plant growth. Moreover, increase in root length in tomato by adding biochar is also described by Vaccari et al. (2015). Another study reported enhanced root biomass in maize after biochar application (Yamato et al. 2006).

Our findings show that among different concentration of various biochar applied to the potting soil, 3% biochar concentration displayed the most promising results in reducing the nematode attack on plants. Bonanomi et al. (2015) used several concentrations of biochar against few soil borne diseases and found 3% as the most conducive treatment to control the studies plant diseases. However, in another study from Huang et al. (2015), 1.2% was concluded the best concentration in potting medium for controlling M. graminicola in rice plants. Recently, addition of nutshell biochar displayed reduction of Meloidogyne spp. population, egg masses and galls in addition to a significant increase in the overall performance of tomato plant (Ibrahim et al. 2018) and these findings support our results.

Our results showed that number of galls and females in roots were higher and plants showed partial wilting in positive control (only nematodes) and they exhibited strongly negative correlation as shown in (Fig. 4E–F). This was in accordance with the results of Mitkowski and Abawi (2003) who reported stunted growth in lettuces due to dense galling by Meloidogyne spp. Our results showed that while increasing in number of females and galls had reduced the fresh shoot and root weight of the plant (Table 1) and (Fig. 4C–D) also agreed with the findings of Maleita et al. (2012) who concluded that plants which receive heavy inoculum exhibits stunted growth and yield. All three biochars treatments had significantly reduced number of egg masses as compare to control. Regression analysis showed (Fig. 4G) that number of females and root length were negatively correlated and it was in accordance with the report of Sharma and Sharma (2015), who indicated decrease in root length as a result of RKN infection in tomato plants. It has been studied (Anwar and Gundy 1993), that root weight increased due to penetration and formation of huge galls on the roots (Fig. 4C–D), which conforms to the present study in which root weight increased due to increasing the number of galls.

Enhancement of nematode resistance in biochar treatments could be due to number possible reasons. For instance, biochar amendment may increase soil pH that could lead to reduce the nematode population (Novak et al. 2009). Moreover, increase in soil pH led to the reduction of number of galls and egg masses with increasing the rate of biochar in potting medium (Ibrahim et al. 2018). Similarly, addition of biochar may accumulate beneficial microbes which could be helping to enhance the plant growth in addition to nematode control through antagonistic effects of these microbes against nematodes. Another important explanation could be the formation of phytoliths in plant roots due to high concentration of silica in all of the biochars. This phytolith formation provides physical barrier in the form of depositions in the cell wall matrix to hinder the entry of nematodes into the plant roots (Alhousari and Greger 2018). Moreover, defense response could be induced by silicon present in biochars to produce several enzymes which possibly can inhibit nematode establishment on the plant roots as observed in cucumber against Pythium spp.

Conclusion

Potential of biochar in management of root knot nematode was confirmed by overall increment in plant biomass and reduction in nematode attack in biochar amended soil. Whereas the study revealed that the appropriate biochar among the three is 3% w/w Rice husk biochar in comprising the plant growth and reduction in nematode infection. Moreover, biochar has no direct nematicidal effect on root knot nematode. Reduction in nematode infection through the plant root system is may be due to changing pH of the medium or by producing ammonia compound in soil as result of degradation of biochar (Ibrahim et al. 2018). Through SEM analysis it has analyzed that the biochar surface is the best habitat for rhizosphere organisms (mostly beneficial) they may involve direct or indirectly to inhibit the nematode invasion in root zone. Biochar particles may resist in the locomotion of fast-moving nematodes between the soil particles. Moreover, the presence of silica compounds in biochar’s may directly involve inhibiting the nematode penetration in root system. Although, SCB biochar showed no promising results in the management of root knot nematodes but somehow it contributed in enhancing overall plant growth.

Acknowledgments

The authors are highly thankful to Higher Education Commission of Pakistan for partially supporting this research through Pak-Turk Researcher’s Mobility Grant No. 9-5(Ph-1-MG-2) Pak-Turk-R&D-HEC-2017.

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