*2.11. Statistical Analysis*

Data were analyzed using SPSS 19.0. software (SPSS Inc. Chicago, IL, USA) and statistical significance was calculated using a one-way analysis of variance (ANOVA). The means of different parameters for each treatment group were compared among each other using a Fisher's protected least significant difference (LSD) test at *p* < 0.05. All figures for statistical analyses were made using Sigma Plot 10.0 (SPSS Inc., Chicago, IL, USA).

#### **3. Results**

### *3.1. E*ff*ect of Fermentation Filtrates on Hatching of Meloidogyne Incognita Eggs*

The fermentation filtrate of *A. japonicus* ZW1 at various concentrations and different time points showed significant nematicidal activity against cumulative hatching rate of eggs. The cumulative hatching rate of eggs increased over time in the 1-WF, 2-WF, and 3-WF treatments (Figure 1). In relative comparison to 1-WF, *M. incognita* eggs exhibited higher sensitivity to 2-WF and 3-WF. Fifteen days after incubation, the cumulative hatching rates in 20% and 50% 1-WF were 71.1% and 30.1%, respectively, and were significantly lower in comparison to 2.5%, 5%, and 10% 1-WF and controls (*p* < 0.05). For the 2-WF treated samples, cumulative hatching rates in 5%, 10%, 20%, and 50% 2-WF were 42.5%, 36.0%, 24.3%, and 6.4%, respectively, 15 d after incubation. These values were significantly lower than that of the 2.5% 2-WF and control treatments (*p* < 0.05). Cumulative hatching rates in 5%, 10%, 20%, and 50% 3-WF treatments were 53.0%, 42.2%, 34.6%, and 21.2%, respectively, 15 d after incubation. These results were significantly lower than that of the 2.5% 2-WF and control treatments (*p* < 0.05).

**Figure 1.** Cumulative *Meloidogyne incognita* eggs hatching rates in *Aspergillus japonicus* ZW1 fermentation filtrate. The bars represent the standard error. The same letter is not significantly different (*p* < 0.05) according to a Fisher's protected least significant difference (LSD) test.

#### *3.2. Nematicidal Activity of Fermentation Filtrates on Meloidogyne Incognita J2s*

The time of culturing influenced the nematicidal activity of the fermentation filtrate on J2s (Figure 2). In comparison to the 1-WF and control treatments, the mortality of J2s was higher in 2-WF and 3-WF treatments at different time points post incubation. In the 1-WF treatment, the mortality of J2s was less than 3.3% and no significant difference was observed after treatment for a 6 to 48 h period. Conversely, application of 2-WF and 3-WF resulted in a significantly higher mortality of J2s at different concentrations of the fermentation filtrates as compared to the controls (*p* < 0.05). When investigating 50% 2-WF and 3-WF, the mortality of J2s reached 100% after a 6 h incubation period. After the 48 h incubation period, the mortality of 2.5% 2-WF and 3-WF treatments reached 56.1% and 56.8%, respectively, and were all significantly higher than the controls (*p* < 0.05). From a morphological perspective, treatment with 2-WF resulted in differences in the J2 when compared to the controls (Figure 3). Specifically, microscopic observations revealed that the bodies of J2s in the 2-WF treatment were either straight or arched without movements at 6 h post-incubation (Figure 3, A2). However, bubbles (Figure 3, Bu) appeared in the body of J2s over time and protruded wrinkles (Figure 4, Wr) on the body surface and areas of intensive cytoplasmic vacuolization were observed (such as damaged areas; Figure 5, Da) at 10 h post-exposure to treatment with 2-WF.

**Figure 2.** The mortality of *Meloidogyne incognita* J2s in *Aspergillus japonicus* ZW1 fermentation filtrate. Means with the same letter in each group designate no significant differences (*p* < 0.05) based on analysis with a Fisher's protected LSD test.

#### *3.3. Greenhouse Experiment*

Treatment with fermentation broth of *A. japonicus* ZW1 resulted in a significant reduction in the number of root galls and eggs per plant as compared to controls (Table 1). The number of root galls and eggs were 8.2 and 3488.9 per plant in the 50% fermentation broth treatment, respectively; whereas 16.8 and 6020 were observed per plant in the 20% fermentation broth treatment, respectively. In both treatments, the number of root galls and eggs was significantly lower than what was observed in controls (*p* < 0.05). The 50% fermentation broth decreased root galls by 78.6% and eggs by 69.4% per plant in comparison to treatment with the Czapek medium control (38.4 root galls and 11413.3 eggs) and 79.9% root galls and 72.0% eggs per plant compared with the tap water control (40.8 root galls and 12480.0 eggs, respectively), and root galls and eggs from the 20% fermentation broth treatment decreased by 56.3% and 47.3% per plant compared with the Czapek medium control (38.4 root galls and 11413.3 eggs, respectively), and 58.8% and 51.8% compared with the tap water control (40.8 root galls and 12480.0 eggs, respectively).

**Figure 3.** Morphology of second-stage juveniles of *Meloidogyne incognita* treated with 10% 2-week fermentation filtrate (2-WF) of *Aspergillus japonicus* ZW1. **A1**–**A4** were treated with 10% 2-WF; **B1**–**B4** were treated with sterilized water; **A1** and **B1** were treated at 0 h; **A2** and **B2** were treated at 6 h; **A3** and **B3** were treated at 12 h; and **A4** and **B4** were treated at 24 h. **Bu**: bubbles. Scale bars of **A1**–**A4** and **B1**–**B4** were 100 μm.



Values represent means ± standard error of six replicate plants per treatment using the combination of two different experiments. Means with the same letter were not significantly different (*p* < 0.05) according to a Fisher's protected LSD test.

**Figure 4.** Visualization of the effect of 10% 2-WF of *Aspergillus japonicus* ZW1 on the morphology of *Meloidogyne incognita* J2s with scanning electron microscopy. (**A**,**C**,**E**) J2s treated with 10% 2-WF. (**B**,**D**,**F**) J2s treated with sterilized water. (**A**,**B**) Head region of J2. (**C**–**F**) The lateral field of J2. Scale bars of (**A**,**B**,**E**,**F**) and (**C**,**D**) were 2 and 5 μm, respectively. **Wr**: protruded wrinkles (black arrow).

**Figure 5.** Cross-sections of *Meloidogyne incognita* J2 treated with 10% 2-WF of *Aspergillus japonicus* ZW1. (**A**–**C**) J2s treated with *A. japonicus* ZW1 fermentation filtrate. (**D**–**F**) J2s treated with sterilized water. Scale bars of **A**, **B**, **C**, **D**, **E**, and **F** were 2 μm. **Da**: damaged and area. **Gu**: gut. **Dn**: destructed nuclei.

#### *3.4. E*ff*ect of Boiling and Storage Time on the Nematicidal Activity of Fermentation Filtrate*

The mortality of J2s in fresh and boiled 10% 2-WF did not display any significant differences (Table 2). After a 48 h incubation period, the mortality of J2 reached 100.0% in both fermentation filtrates and was significantly higher than what was observed in the sterilized water treatment (*p* < 0.05).

**Table 2.** Nematicidal activity of the boiled fermentation filtrate of *Aspergillus japonicus* ZW1 on *Meloidogyne incognita* J2s.


Values represent means ± standard deviation of three replicates. Means with the same letter are not significantly different (*p* < 0.05) according to a Fisher's protected LSD test.

No significant difference was observed in the mortality of J2s exposed to different storage conditions of 10% 2-WF (Table 3). Specifically, they all reached 100% mortality after a 48 h incubation period, which was higher than the sterilized water treatment (*p* < 0.05).


**Table 3.** Mortality of *Meloidogyne incognita* J2s in *Aspergillus japonicus* ZW1 fermentation filtrate under different storage conditions.

Values represent the means ± standard error of four replicates; means with the same letter are not significantly different (*p* < 0.05) according to a Fisher's protected LSD test.

#### *3.5. E*ff*ect of Fermentation Filtrate on Seed Germination*

The 20% and 10% 2-WF did not influenced the germination of corn, rice, tomato, cowpea, and cucumber seeds (Table 4). Two days after incubation with 10% 2-WF, the wheat seed germination rate was 85.4% and was significantly higher than what was observed in the control (*p* < 0.05). After an extended period of time beyond the 48-h time period, this value did not increase any further. For soybean seeds treated with 10% 2-WF, germination was significantly lower than what was observed in sterilized water (*p* < 0.05) at day 1; however, there were no statistically significant differences 2–5 days post-incubation across 20% and 10% 2-WF and sterilized water treatments. For cabbage seeds, germination in 20% 2-WF was significantly lower than what was observed in 10% 2-WF and control treatments (*p* < 0.05).


**Table 4.** Seed germination (%) in different concentrations of 2-week *Aspergillus japonicus* ZW1 fermentation filtrate.

Values represent the means ± standard error of four replicates; means with the same letter were not significantly different (*p* < 0.05) according to a Fisher's protected LSD test.

#### *3.6. Structural Confirmation of Nematicidal Substance from 2-WF*

The active compound was a pale-yellow crystal, which can dissolve easily in water. The 1H NMR spectrum in MeOH exhibited signals due to two methyl groups at δ 3.68 (each 3H, s, 7, 8-CH3), 2.95, 2.85 (each 2H, AB system, d, *J* = 12.0 Hz, 2, 4-CH2). The 13C NMR and heteronuclear multiple-quantum correlation spectra revealed two carbonyl carbons at δ<sup>C</sup> 175.00 (s, C-6), 170.46 (s, C-1, C-5), two methoxy groups 72.84 (s, C-3), 50.78 (q, C-7, C-8), 42.63 (t, C-2, C-4). The electrospray ionization mass spectrometry (ESI-MS) data of active compound was identified the molecular formula of C8H12O7 by the [M]<sup>−</sup> ion signal at *m*/*z* 219 [M]−. The structure of the active compound was determined to be 1,5-Dimethyl Citrate hydrochloride ester (C8H12O7 HCl, Figure 6) by the analysis of its spectroscopic data and comparison with the values in the literature [35].

**Figure 6.** Chemical structures of active compound from *Aspergillus japonicus* ZW1 fermentation filtrate.

#### *3.7. E*ff*ect of 1,5-Dimethyl Citrate Hydrochloride Ester on Meloidogyne Incognita J2s*

1,5-Dimethyl Citrate hydrochloride ester had a strong toxic activity against J2s at low concentrations, and J2s mortality increased with the duration of exposure in different concentration of 1,5-Dimethyl Citrate hydrochloride ester (Table 5). There were significant differences in mortality between concentrations and control after exposure (*p* < 0.05). The mortality of J2s in concentrations of 1.25, 1.00, 0.75, 0.50, and 0.25 mg mL−<sup>1</sup> of 1,5-Dimethyl Citrate hydrochloride ester were 91.7%, 57.7%, 36.9%, 20.8%, and 3.3% respectively at 48 h after exposure, which were significantly higher than that of sterilized water (*p* < 0.05).


**Table 5.** Mortality (%) of *Meloidogyne incognita* J2s in different concentrations of active compound from *Aspergillus japonicus* ZW-1 fermentation filtrate.

Values represent the means ± standard error of four replicates; means with the same letter each column were not significantly different (*p* < 0.05) according to a Fisher's protected LSD test.

Nematicidal activity of 1,5-Dimethyl Citrate hydrochloride ester was evaluated by comparing the median lethal concentrations (LC50) for different concentrations on *M. incognita* J2s under different exposure times. The concentrations at which 50% of the dead *M. incognita* J2s (LC50) were 1.0373, 0.9646, 0.9397, and 0.7614 mg mL−<sup>1</sup> 1,5-Dimethyl Citrate hydrochloride ester for 6, 12, 24, and 48 h respectively. The LC50 values were decreasing with the enhanced of exposure time (Table 6).



LC-lethal concentration expressed in mg/mL active compound with 95% confidence intervals (CI). SE, standard error.

#### **4. Discussion**

In general, the management of parasitic nematodes is a challenging process and current control strategies are mostly dependent upon the application of nematicides [36]. However, many effective nematicides have been restricted for usage and have been banned from the market in recent years due to environmental concerns [37]. Biological options are gaining attention as promising new tools due to their environmentally-friendly and non-toxic characteristics. The potential for using microbes in controlling plant-parasitic nematodes has been documented [38] and effective microbes have been obtained from soil, plants, and the surface of nematodes [39–41]. *Aspergillus* spp. are very common in soil and are lethal to the nematode population; *A. niger* and *A. candidus* were the potential fungal agents to be used against plant-parasitic nematodes [35,42,43]. The results of this study indicated that fermentation of the *A. japonicus* ZW1 from soil was found to not only inhibit egg hatching but was also toxic to nematodes in vitro. The 2-WF was shown to be more toxic to J2s than 1-WF and 3-WF; this effect showed the presence of more active compounds in 2-WF, worth previous characterization. The similar behavior of several fungi and bacteria were also studied against plant parasitic nematodes. Among them a culture filtrate of the rhizosphere bacterium *Pseudoxanthomonas japonensis* isolated from soil exhibited strong nematicidal activity against the *M. incognita* [30]; a metabolite of *Xylaria grammica* KCTC 13121BP isolated from lichen showed strong J2 killing and egg-hatching inhibitory effects [44];

and a culture medium of *Stenotrophomonas maltophilia* and *Rhizobium nepotum* isolated from the surface of nematodes reduced the pathogenicity of wild pine wood nematodes [39].

Natural products have many limitations, such as natural laccases, which have poor stability of enzymatic activity [45]. As a result, it was important to determine and assess if the novel environmentally-friendly nematicides could be stable for practical and durable application opportunities. Consequently, in our present study, we were interested to determine the durability of the novel biological filtrates. Importantly, the toxic activity of the *A. japonicus* ZW1 fermentation filtrate was not effected by boiling, storage time (1-, 2-week, and 3-week) and warm/cold conditions (25 ◦C and 4 ◦C). Usually, the surface coating of nematodes was considered to play an important role in the external protection of nematode bodies, sensing, and communication [46,47]. The microbes and plant produced several acidic metabolites or proteinases that specifically degraded the outer membrane of host cells during primary infection [42,48,49]. In our study, wrinkles on the surface of the body of J2s in 2-WF were observed with scanning electron microscopy, and internal bubbles appeared in their body over time. Additionally, other prominent changes such as intensive cytoplasmic vacuolization areas were observed using transmission electron microscopy; suggesting that the activity of compounds produced by *A. japonicus* ZW1 targeted the skin of nematodes and changed its permeability [50]. Previous research showed acidoid (acetic acid) damage the nuclei of cells and led to intensive cytoplasmic vacuolization areas in the body of J2 *M. incognita* [28]. Nematicidal metabolites from the endophytic fungus *Chaetomium globosum* YSC5 significantly reduced the reproduction of *M. javanica* as well [51]. In our present study, nematicidal compound 1,5-Dimethyl Citrate hydrochloride ester from *A. japonicus* ZW1, first isolated and identified on the basis of NMR, LC-MS techniques, was different with the nematicidal compounds produced by *A. niger* (oxalic acid) and *A. candidus* (Citric acid and 3-hydroxy-5-methoxy-3-(methoxycarbonyl)-5-oxopentanoic acid). *M. incognita* J2 mortality reached 100% at 1 day, and egg hatching was suppressed by 95.6% at 7 days after treated with 2 mmol L−<sup>1</sup> (180 μg mL<sup>−</sup>1) oxalic acid [42]. 3-hydroxy-5-methoxy-3-(methoxycarbonyl)- 5-oxopentanoic acid was an isomer of 1,5-Dimethyl Citrate, which increased the mean percentage of immobile *Ditylenchus destructor* by 50% at a concentration of 50 mg mL−<sup>1</sup> after exposure for 72 h [35]. In our study, *M. incognita* J2 treated with 1,5-Dimethyl Citrate hydrochloride ester, mortality reached 91.7% at 48 h after exposure to 1.25 mg mL−<sup>1</sup> concentration, the LC50 was 0.7614 mg mL<sup>−</sup>1, which exhibited the most potent toxic activity against the J2 of *M. incognita*. However, the interesting thing was that in in vitro bioassay, fermentation of the strain exhibited better nematicidal effects, and the mortality of J2s reached 100% after exposed to 5% concentration (approximately 100 μg mL−<sup>1</sup> 1,5-Dimethyl Citrate hydrochloride ester) *A. japonicus* ZW1 fermentation filtrate at 24 h. Our speculation is that the nematicidal effect originated 1,5-Dimethyl Citrate hydrochloride ester combined with some other compounds produced by *A. japonicus* ZW1. Thus, we still need further study to find and proved other nematicidal activity compounds by metabonomics analysis.

No effect on the seed germination of corn, wheat, rice, cowpeas, cucumbers, soybeans, and tomatoes was observed for the 10% and 20% 2-WF treatments. In whole pot experiments, treatment with the fermentation broth of the strain suppressed root galls and egg populations for tomatoes. As a result, these results suggested that *A. japonicus* ZW1 produced and excreted metabolites that were toxic to root-knot nematodes but did not exert negative effects on seed germination. Thus, *A. japonicus* showed desirable, effective, and safe biocontrol properties against *M. incognita* for both in vitro and greenhouse conditions. Taken together, these observations suggest that the fermentation filtrate of *A. japonicus* ZW1 is safe for use as a biological control fungus against root-knot nematodes. However, further studies are warranted and necessary to evaluate the in vivo efficacy of the strain against root-knot nematodes or other plant-parasitic nematodes.

#### **5. Conclusions**

*A. japonicus* ZW1 fermentation filtrate exhibited a potential biocidal activity on *M. incognita* in vitro and in vivo. The *A. japonicus* ZW1 2-week fermentation filtrate exhibited markedly inhibitory effects on egg hatching and nematicidal activities on J2s followed by 3-week fermentation filtrate. The *A. japonicus* ZW1 filtrate penetrated the body wall of *M. incognita* and caused intensive cytoplasmic vacuolization with remarkable protruded wrinkles appearing on the body surface of the J2s. Moreover, the nematicidal activity of the fermentation was stable after a boiling treatment and was not affected by storage time. *A. japonicus* ZW1 fermentation filtrate had no negative effect on the viability and germination of corn, wheat, rice, cowpeas, cucumbers, soybeans, and tomato seeds. The main active compound of 1,5-Dimethyl Citrate hydrochloride ester was first isolated and identified from the *A. japonicus* ZW1 fermentation filtrate. Finally, this work highlights the relevance of *A. japonicus* ZW1 fermentation filtrate as a potential new biological nematicide resource for the control of *M. incognita*.

**Author Contributions:** Conceptualization, Q.H., A.M. and H.W.; methodology, Q.H., A.M.; software, D.W.; B.L. and A.M.; validation, Q.H.; formal analysis, Q.H., A.M., D.W. and B.L.; investigation, A.M. and H.W.; resources, H.W.; data curation, Q.H., A.M. and B.L.; writing—original draft preparation, Q.H.; writing—review and editing, Q.H., A.M., D.W., and H.W.; and supervision, H.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the National Natural Science Foundation of China (31660511, 31460464), Guangxi Innovation Team of National Modern Agricultural Technology System (nycytxgxcxtd-10-04), Construction Project of Characteristic Specialty and Experimental Training Teaching Base (Center)-Characteristic Specialty-Plant Protection (2018–2020).

**Conflicts of Interest:** All authors declare they have no conflict of interest.
