Control of A Tomato Plant Root-Knot Nematode By Induced Resistance Of Oxalic Acid Derived From Aspergillus Niger

Aspergillus niger F22 producing oxalic acid (OA) as a nematicidal component is currently used as a microbial nematicide. OA is known to induce systemic resistance in plant diseases caused by fungi, bacteria, and viruses, but the induced resistance of OA has not yet been elucidated in plant diseases caused by root-knot nematodes (RKNs). In this study, we investigated the functional mechanism of induced resistance of A. niger F22 formulation (Nemafree, 20% SC) and OA in tomato plant RKN disease caused by Meloidogyne incognita and analyzed their effectiveness against the disease. Foliar spray and soil drench treatments of Nemafree and OA were effective in the management of M. incognita in tomato plant in-pot experiments. When Nemafree and OA were applied 4 days before inoculation of M. incognita eggs, the treatments of Nemafree (4,000-fold dilution) and OA (0.22 mM) reduced root gall formation by more than 50%. The soil drench treatment also effectively suppressed RKN disease in eld experiments. Moreover, the treatments of Nemafree and OA enhanced the transcriptional expression of pathogenesis-related 1 gene, plant proteinase inhibitor-II, and polyphenol oxidase genes and improved the production of total phenols, avonoids, and lignin in the tomato plants infected with M. incognita. These results demonstrate that RKN diseases can be effectively controlled by induced resistance even at low concentrations of Nemafree or OA. Accordingly, our study provides evidence for more economical and ecient application strategies of microbial nematicides that control RKNs under eld conditions.


Introduction
Root-knot nematodes (RKNs; Meloidogyne spp.) are a group of plant-parasitic nematodes (PPNs) responsible for marked economic damage by causing diseases to various plants worldwide (Kayani Plant diseases caused by RKNs are more di cult to predict and control than those by other microorganisms because of the short generation time and high regeneration rate of RKNs in soils However, we predicted that active organic acids contained in a formulation of active microorganisms could solve this problem in soil. A previous study reported that Aspergillus niger F22 strain produces OA as a signi cant nematicidal metabolite and shows su cient control of RKNs in vitro and in the eld by exhibiting direct nematicidal activity (Jang et al. 2016). This effect led to the development of a commercial microbial nematicide called Nemafree (A. niger F22 20% SC, Farm Hannong Co., Seoul, Korea).
The control of RKN disease with organic acids has been studied for many years ( improve defense against RKN disease in eld conditions. Therefore, we aimed to verify whether the RKN could be controlled by induced plant resistance elicited by Nemafree and the active component OA itself. The objectives of this study were 1) to determine whether Nemafree and its active component OA could induce resistance to M. incognita for effective disease management in tomato plants, 2) to con rm the induction of resistance against M. incognita by enhanced expression of defense-related genes and secondary metabolites, 3) to evaluate the in vivo e cacy of Nemafree by induced resistance and direct nematicidal activity against RKN disease, and 4) to propose the optimal application strategies of commercial microbial nematicides for the management of RKN disease in eld conditions.

Materials And Methods
Nematodes Root-knot nematodes (Meloidogyne incognita) were isolated and identi ed by the Korea Research Institute of Chemical Technology (Daejeon, Korea). They were maintained on tomato plants (Solanum lycopersicum Mill. cv. Seogwang) in a greenhouse at 25 ± 5°C with day/night lighting of 16 h/8 h. At 8 weeks after infection with M. incognita, the infected tomato plants were uprooted and gently washed with tap water to identify gall formation on the roots. The roots were then cut into 1 cm pieces and homogenized in a blender (HM-2100S; Hanil, Korea) in a 1% sodium hypochlorite (NaOCl) solution at 14,500 rpm for 1 min. Eggs of M. incognita were collected using 45 µm and 25 µm sieves. The collected eggs were rinsed with distilled water and used for in vivo pot experiments.

Oxalic acid analysis
The content of oxalic acid produced by A. niger F22 in a commercial microbial nematicide Nemafree (A. niger F22 20% SC, Farm Hannong Co.) was measured using an HPLC system (Waters Alliance e2695 system, Milford, Massachusetts, USA) at 30°C using an Aminex HPX-87H column (300 mm × 7.8 mm, Bio-Rad, Hercules, CA, USA). Elution was carried out isocratically using 5 mmol/L sulfuric acid. The ow rate and detection wavelengths were 0.6 mL/min and 210 nm, respectively. Nemafree diluted 100-fold with water was ltered through a 0.45 μm PTFE syringe lter (Whatman, USA) and injected into the HPLC system during the mobile phase ow. Quantitative analysis of OA was performed using standard curves.

RNA extraction and RT-qPCR analysis
For the analysis of defense-related gene expression, tomato plants were treated with Nemafree (4,000fold dilution) and OA (0.22 mM) by soil drench. Roots were collected 1, 2, and 4 days after treatment and 2, 4, and 8 days after inoculation. Roots were ground in liquid nitrogen using a pestle and mortar. According to the manufacturer's recommendations, total RNA was extracted using IQeasy™ Plus Plant RNA Extraction Mini Kit (iNtRON Biotechnology, Seong-nam, Korea). Qualitative and quantitative analyses of all RNA samples were performed using a Nano-drop spectrophotometer (NANOPhotometer® NP80, Implen, Munich, Germany). cDNA libraries were synthesized from total RNA using oligo (dT) primers and SuperScript™ IV reverse transcriptase (Invitrogen Inc., Carlsbad, CA, USA), following the manufacturer's instructions.
The PCR primers (Table 1)   The cDNA libraries were ampli ed in real-time quantitative PCR using iQ™ SYBR Green supermix (Bio-Rad Laboratories, Hercules, CA, USA) following the manufacturer's instructions. The PCRs were conducted using a real-time PCR detection system (Bio-Rad CFX 96; Bio-Rad Laboratories). The results were analyzed using the BioRad CFX Manager Version 2.1. Relative fold changes in gene expression levels between treatments were calculated using the △△CT method after normalizing the levels to those of the reference gene UBI. All tests were repeated three times.

Quantitative analysis of secondary metabolites
To analyze the secondary metabolites related to the plant defense system, tomato plants were treated with Nemafree (4,000-fold dilution) and OA (0.22 mM), and inoculated with 10,000 M. incognita eggs 4 days after treatment. The plant roots were then collected 2, 4, 8, and 14 days after inoculation.
Total phenolic, avonoid, and lignin contents were analyzed according to Deng et al. (2015). Tomato root samples (1 g) were then homogenized in 5 mL of 1% ice-cold HCl-methanol solution for 2 h. The homogenized material was then centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant was used to quantify the total phenolics and avonoids. The total phenolic content was measured at 280 nm using gallic acid (purity ≥ 97.5, Sigma Aldrich) as a standard. The avonoid content was measured at 325 nm using quercetin (purity ≥ 95%, Sigma Aldrich) as a standard.
Lignin content was determined using a homogenized tomato root sample (1 g) in 4 mL of 95% ice-cold ethanol solution. This sample was centrifuged at 12,000 rpm for 10 min at 4°C. After centrifugation, the pellets were collected, washed three times with 95% ethanol, and then rewashed three times with ethanolhexane (1:2, v/v) solution. The pellets were dried overnight in an oven at 47°C and transferred to a new tube, where 1 mL of 25% bromized acetyl-acetic acid was added and then incubated for 30 min at 70°C. The reaction was stopped by adding 1 mL of 2 M NaOH to each tube. Then, 2 mL of ice-cold acetic acid and 0.1 mL of 7.5 M hydroxylamine hydrochloric acid were added, followed by centrifugation at 12,000 rpm for 15 min at 4°C. The supernatant was used to measure lignin content, which was quanti ed at 280 nm using lignin (Sigma Aldrich) as a standard.

Field experiment
From June to August 2020, eld experiments were conducted at a tomato greenhouse farm (Jungjeong-ri, Buyeo, Chungnam, Korea) naturally infected with Meloidogyne spp. Tomato seeds (Solanum lycopersicum Mill. cv. TY Nonari) were planted in a horticulture nursery medium on seed trays in a greenhouse and then transplanted into a plot after 3 weeks. The eld experiments used a randomized block design with ve replicates, and each treatment consisted of a 10 m 2 plot (2 × 5 m 2 ), each containing 15 tomato plants.
The three eld treatment methods were (1) untreated control, (2) A. niger F22 formulation Nemafree (A. niger F22 20% SC, 4,000-fold dilution) and (3) soil incorporation using chemical nematicide Sunchungtan (fosthiazate 5% GR, Farm Hannong Co.). Seven days before transplantation, the tomato seedlings were treated once with Nemafree. After transplantation, four treatments with Nemafree were performed at 10day intervals by soil drench. The untreated control was watered by soil drench four times at 10-day intervals. For the positive control of chemical nematicide, Sunchungtan was mixed with soil at a concentration of 6 g/m 2 before transplantation. All tomato roots were collected 75 days after the rst treatment of Nemafree, and their gall formation was con rmed using the GI as in the pot experiments.
Tomato rhizosphere soil was also sampled from each plot 10 days before and 75 days after the rst treatment to determine the density of nematodes in the eld soil. The number of nematodes per 100 cm 3 of tomato rhizosphere soil was analyzed using the Berman funnel method (Huang et al. 2014;Jenkins 1964).

Statistical analysis
The parameters measured in this study were designed to evaluate the induced resistance activity and e cacy of Nemafree and OA against RKN disease. The analyses were conducted separately for in-pot and eld experiments. All data were analyzed for homogeneity of variance using SPSS statistical analysis software (version 21.0 for Windows; SPSS, Chicago, IL, USA). The data are expressed as means ± standard deviations of replicates and evaluated by one-way analysis of variance (ANOVA). Statistical differences among treatments were determined using Tukey's multiple-range test (p < 0.05).

Analysis of OA in A. niger F22 formulation Nemafree
OA is the nematicidal metabolite produced by the A. niger F22 strain; we predicted that OA induces resistance to RKNs in tomato plants. Therefore, the content of OA in a commercial microbial nematicide Nemafree (A. niger F22 20% SC, Farm Hannong Co.) was analyzed to determine the optimal concentration of Nemafree and OA for this study. The retention time of OA was 6.67 mins; we con rmed Nemafree contained about 0.88 M of OA using the formula (y = 11843x − 3E + 06) obtained from the standard curve (Fig. 1).

Effect of Nemafree and OA on tomato plants and nematode infection
Since the infestation of M. incognita occurs at the plant roots in soils, we hypothesized that the direct nematicidal activity of Nemafree or OA would not work if tomato plant leaves were treated with Nemafree or OA by foliar spray only, without permeation to the soil or root area. Therefore, we analyzed the e cacy of pretreatment of Nemafree and OA by foliar spray and soil drench 4 days before M. incognita inoculation in the pot experiments. In addition, through pretreatment at various concentration of Nemafree and OA, we investigated the optimal concentration of Nemafree and OA treatments for the management of RKNs. As the undiluted Nemafree (A. niger F22 20% SC) contained 0.88 M of OA, 1000fold diluted Nemafree was equivalent to 0.88 mM OA.
Six weeks after inoculation of M. incognita, foliar spray of Nemafree and OA repressed root gall formation and egg mass production at most concentrations used in the experiment (Fig. 2). The treatment with the diluted Nemafree (1,000 ~ 4,000-fold) and OA (0.44 ~ 0.88 mM) on tomato plant leaves resulted in a signi cant decline of root gall formation in the infected plants, and their control values reached up to 40.91 ~ 43.18%, considering that the mode of action of Nemafree and OA is independent of direct nematicidal activity (Fig. 2a, b). Similar to the result of root gall formation, treatment with the diluted Nemafree (2,000 ~ 4,000-fold) and OA (0.44 ~ 0.88 mM) by foliar spray decreased egg mass production in the infected plants, with control values of 24.88 ~ 42.24% (Fig. 2c, d).
In contrast, when 5,000-fold diluted Terranova (the commercial chemical nematicide, Abamectin 1.68% SC), a positive control with direct nematicidal activity, was treated by foliar spray 4 days before M. incognita inoculation under the same conditions as Nemafree and OA treatments, it was con rmed that there was no effect at all, resulting in a similar root galling index (3.33) and egg mass production (39.14) to the untreated control (3.83 and 41.15, respectively) (Fig. 2).
Soil drench of Nemafree and OA also inhibited root gall formation and egg mass production at most concentrations used in the experiment, and their e cacy against M. incognita by soil drench was slightly higher than that by foliar spray (Fig. 3). Treatment with the diluted Nemafree (4,000 ~ 8,000-fold) and OA (0.22 mM) by soil drench effectively suppressed root gall formation in the infected plants, with control values of 52.25 ~ 63.64% (Fig. 3a, b). As expected, when 5,000-fold diluted Terranova (Abamectin 1.68% SC) was applied by soil drench, it had control values of 100% and 95.73% for the inhibition of root gall formation and egg mass production, respectively. Although the control value of Nemafree and OA on the inhibition of egg mass production was lower than that of the positive control abamectin, treatment of Nemafree and OA (except 0.88 mM OA) by soil drench reduced egg mass production in infected tomato plants by 41.00 ~ 66.88%, showing excellent nematicidal activity (Fig. 3c, d).
Taken together, the formation of root galls and egg masses on tomato plant roots was reduced by treatment with Nemafree and OA using both foliar spray and soil drench ( Fig. 2 and Fig. 3). The e cacy of Nemafree and OA in controlling RKN disease was dose-independent. When compared with the positive control Terranova (Abamectin 1.68% SC), which had no disease control e cacy against RKNs by foliar application, Nemafree and OA had induced resistance and direct nematicidal activity against RKNs on PR-1 was induced < 2-fold at 8 days after nematode inoculation compared to that with no inoculation (Fig. 4a); however, the induction at 8 days after inoculation was consistent and increased signi cantly in every replicate. Further, PR-1 was induced 3 to 5-fold in the tomato plant roots by Nemafree treatment and 1.41 to 2.78-fold by OA when compared to that in the untreated tomato roots (Fig. 4a). PI-II was induced approximately 10-fold by nematode inoculation and approximately 8-fold and 6-fold by Nemafree and OA treatment, respectively (Fig. 4b). However, PI-II was only induced 2 days after treatment with Nemafree and OA and then returned to normal expression levels. ERF1 showed an opposing pattern to PR-1 and PI-II. The nematode inoculation and both Nemafree and OA treatments repressed ERF1 expression (Fig. 4c).
The ROS response to nematode inoculation was complex, unlike the other genes. PPO was induced approximately 4-fold and 3-fold higher than that in the untreated samples at 2 days and 8 days after inoculation, respectively, but was suppressed at 4 days after inoculation (Fig. 4d) We concluded that the tomato plants pretreated with A. niger F22 formulation Nemafree (4,000-fold dilution) and OA (0.22 mM) induced resistance to M. incognita through activation of the SA and JA signaling pathways involved in the plant defense system. This further suggests that the crosstalk between the SA and JA-related pathways and ROS is closely related to the defense response against RKN infection in tomato plants.
Effects of Nemafree and OA on total phenolic, avonoid, and lignin production in tomato roots The production of secondary metabolites related to the plant defense system was analyzed at 2, 4, 8, and 14 days after inoculation of M. incognita eggs into tomato plant roots. From 4 days to 14 days after inoculation of the eggs, total phenolic content in Nemafree (4,000-fold dilution)-and OA (0.22 mM)treated plants increased and was maintained, unlike that in the untreated plants (Fig. 5a). At 8 days after inoculation, the total phenolic content in the roots treated with Nemafree and OA increased to 3.35-and 2.64-fold, respectively, compared to that in the untreated roots. The avonoids produced in Nemafree-and OA-treated roots also started to increase at a higher rate than those in the untreated roots from 4 days after inoculation (Fig. 5b). At 4 days after inoculation, avonoid contents in the Nemafree and OA treated roots increased to 1.62-and 1.50-fold, respectively, compared to the those in the untreated plants; the difference was maintained until 14 days after inoculation. Similarly, the lignin content increased signi cantly in Nemafree and OA treated roots from 4 days after inoculation (Fig. 5c). In particular, at 14 days after inoculation, the amount of lignin produced in the Nemafree-and OA-treated plants increased 1.45-fold and 1.52-fold more than that in the untreated plants, respectively.
These results indicate that when tomato roots pretreated with Nemafree (4,000-fold dilution) and OA (0.22 mM) were infected with M. incognita, the production of secondary metabolites (phenols, avonoids, and lignin) related to the plant defense system increased more than that in the untreated controls, from 4 days after inoculation.

E cacy of Nemafree for managing M. incognita in the eld
In tomato elds naturally contaminated with RKNs (Meloidogyne spp.), the population of nematodes in the soil before the rst treatment was 222 ~ 296 nematodes/100 cm 3 . After 75 days of the rst treatment with Nemafree (4,000-fold dilution), the population of nematodes decreased signi cantly to 183 nematodes/100 cm 3 -34.64% less than the initial population-similar to that with the positive control Sunchungtan (26.35% reduction than the initial population). Conversely, in the untreated soil, the population of nematodes increased by 181.08% (402 nematode/cm 3 ) in the same period.
The treatment of Nemafree and Sunchungtan signi cantly lowered the gall index of infected tomato plants in eld conditions and effectively inhibited gall formation by 68.35% and 69.27%, respectively, compared to those of the untreated control group (Table 2). Nemafree (4,000-fold dilution) treatment showed similar e cacy to the chemical nematicide Sunchungtan (fosthiazate 5% GR) in inhibiting gall formation and reducing nematode population in soils.

Discussion
In the previous study, we investigated the disease control e cacy of A. niger F22 strain in eld conditions and its direct nematicidal activity against RKNs (Jang et al. 2016 Radwan et al. (2017) also reported that foliar application at a rate of 10 mM OA was more effective in reducing nematode galls than soil drench application for in-pot experiments of tomato plants infected with RKNs. However, we observed that 0.22 mM of OA, corresponding to Nemafree diluted 4000fold, applied by soil drench rather than by foliar spray, reduced gall and egg mass formation by 63.64% and 66.88% respectively, when compared to those of the untreated plants. This was con rmed in the eld experiments, thus supporting that treatment of 4000-fold diluted Nemafree by soil drench has a similar effect as the chemical nematicide Sunchungtan (fosthiazate 5% GR, Farm Hannong Co.). The e cacy of Nemafree to control RKN diseases was maximized using soil drench treatments in which direct nematicidal activity and resistance-inducing activity in tomato plants act concurrently. This was supported by statistical analyses, which showed that the disease control e cacy of Nemafree in eld conditions was statistically similar to that of the chemical nematicide Sunchungtan, even at low concentrations (4,000-fold dilution), establishing the optimal treatment conditions of soil drench for Nemafree application in the eld.
Plants treated with agents such as non-toxic pathogens, plant extracts, and synthetic chemicals can We also con rmed that secondary metabolites, such as total phenolics, avonoids, and lignin, were  (Table 2). Moreover, at 75 days after the rst treatment, the density of soil nematodes also decreased by 34.64% with Nemafree (4,000fold dilution) and 26.35% with Sunchungtan (5,000-fold dilution), indicating that Nemafree had a highly potent effect on reducing the density of soil nematodes. Nemafree and its active ingredient OA may induce resistance against RKNs through upregulated expression of defense-related genes (PR-1, PI-II, and PPO) and increased production of secondary metabolites (phenol, avonoid, and lignin), consequently enabling tomato plants to tolerate unfavorable conditions and diseases caused by RKNs, as shown in Fig. 6.
This report is the rst study on the management of RKNs in plant diseases using resistance induced by low concentrations of the A. niger F22 formulation Nemafree and OA. In this study, we found that the optimal concentration of the commercial microbial nematicide Nemafree for the management of RKN disease is a 4,000-fold dilution. When treated by soil drench application at a low but optimal concentration (4,000-fold dilution), Nemafree appears to effectively control RKN disease in the eld through induced resistance and direct nematicidal activity. This e cient formulation can be used in elds as a substitute to chemical pesticides to control RKNs and other pathogens. In the elds, Nemafree was applied four times at 10-day intervals by soil drench. Nemafree is eco-friendly and causes less chemical residue or environmental contamination on crops; therefore, it can be applied several times during the planting season. This eco-friendly A. niger F22 formulation can be quickly and conveniently used in a large area using various treatment methods (air spray, drone, etc.) because it is effective at managing RKNs through foliar spray treatment. In this study, we only evaluated the control e cacy of induced resistance by Nemafree and its active component OA on the RKN M. incognita. Therefore, it is necessary to investigate the effect of these microbial nematicides on other nematode species in multiple eld conditions.

Conclusions
Tomato plants treated with A. niger F22 formulation Nemafree (4,000-fold dilution) and OA (0.22 mM) can effectively manage the RKN M. incognita in pot and eld experiments. Treatment with Nemafree and OA induced the expression of PR-1, PI-II and PPO transcripts and enhanced the production of secondary metabolites (total phenolics, avonoids, and lignin). We also recommend the optimal application concentration and strategy of commercial microbial nematodes for RKN disease management in the eld. The microbial pesticides containing OA as an active component can be used as nematicides to sustainably manage RKNs in an eco-friendly way. Soil-borne diseases caused by RKNs can be effectively controlled by induced resistance as well as direct nematicidal activity of A. niger F22 formulation Nemafree and OA, even at low concentrations. We are the rst to identify the possibility of controlling RKN disease by induced resistance using a low concentration A. niger F22 formulation and OA. Based on our results, more economical and e cient application strategies of microbial nematicides can be devised to control RKNs in eld conditions.   Expression of the SA signaling pathway-related gene PR-1 (a), JA signaling pathway-related genes PI-II (b), ET signaling pathway-related gene ERF1 (c), and ROS scavenger-related gene PPO in the roots of tomato plants at 1, 2, and 4 days after treatment (DAT) and before inoculation was detected by qRT-PCR.
OA: Oxalic acid 0.22 mM; NF: Nemafree 4,000-fold dilution; UT: Untreated control. Data are presented as the means ± standard deviation bars of three biological replicates Figure 5 Page 24/25 Total phenolic (a), avonoid (b), and lignin (c) contents of tomato roots treated with Aspergillus niger F22 formulation Nemafree (4,000 fold dilution) and oxalic acid (0.22 mM) at 2, 4, 8, and 14 days after inoculation (DAI) with Meloidogyne incognita eggs. Data are presented as the mean ± standard error bars of three biological replicates. Means with the same letter are not signi cantly different (p < 0.05) according to Turkey's multiple range test Figure 6