**1. Introduction**

Root-knot nematodes (*Meloidogyne* spp.) are economically important worldwide pathogens causing considerable damage to many crops, including cucumbers, tomatoes, rice [1–4], and even cotton [5,6]. *Meloidogyne incognita* is an important species of root-knot nematodes worldwide due to its direct impact on crop yields [7–9]. Specifically, it is capable of causing an estimated yield loss of 5–43% within vegetable crops cultivated in tropical and subtropical areas [10] and estimated \$100 billion loss per year worldwide [11].

Due to their short life cycle and high reproduction rates, these root-knot nematodes have been particularly challenging to control. Previously, chemical nematicides are efficiently used to suppress

nematode populations, such as fenamiphos, sebufos, dazomet, and carbofuran [12]; however, these have been found to be harmful to both the eco-environment and human health due to their toxic effects. Thus, as a result of these negative impacts and the significant economic losses which can result from nematodes, new and alternative biological control options are urgently needed [13]. Therefore, the use of biological agents to suppress the population of plant-parasitic nematodes could provide an alternative strategy to sustainably manage plant-parasitic nematodes. Using biofumigation instead of harmful fumigants (like synthetic nematicide methyl bromide) to control nematodes is an increasingly feasible method of parasitic nematode management [14]. Plants such as *Melia azedarach* have been found to be potential sources of biofumigation plant material to control *Meloidogyne* spp. on tomato [15]. Moreover, microbial agents for the control of plant-parasitic nematodes is also a potential method; such as bacteria [16,17], fungi [18,19] and actinomycetes [20], which are nematophagous or antagonistic for root-knot nematodes. Specifically, *Arthrobotrys irregularis*, *Pochonia chlamydosporium*, *Paecilomyces lilacinus*, *Myrothecium verrucaria*, bacteria *Pasteuria usgae*, *Bacillus firmus*, *Burkholderia cepacia*, *Pseudomonas fluorescens*, and *Streptomyces avermitilis* [21,22] have been commercially used in many countries for the control of plant-parasitic nematodes. Some potential microbial sources were constantly obtained, volatiles from beneficial bacteria (*Bacillus* sp., *Paenibacillus* sp. and *Xanthomonas* sp.) can control *M. graminicola* second-stage juveniles (J2s) on rice and significantly reduced infection of susceptible rice [23]. Co-inoculation of *Streptomyces* spp. strains KPS-E004 and KPS-A032 showed success in suppressing root-knot nematode [24].

In our previous study, *A. japonicus* ZW1 culture filtrate was shown to have marked nematicidal activity against *M. incognita*. As a result, the main objective of this work was to evaluate the potential biological control of *A. japonicus* ZW1 against root-knot nematodes including: (1) the nematicidal activity of *A. japonicus* ZW1 fermentation filtrate on eggs and J2s within pot and in vitro experiments; (2) electron microscopic evaluation of J2 bodies after treatment with 2-week fermentation filtrate (2-WF); (3) effect of boiling and storage time on nematicidal activity stability of the fermentation filtrate; and (4) evaluation for the effect of *A. japonicus* ZW1 fermentation filtrate on the germination of various crop seeds.

#### **2. Materials and Methods**

#### *2.1. Nematode Preparation*

Tomato seeds (cv. Xin Bite 2 F1) were sourced from Yashu Garden Seeds Co., Ltd., (Guangzhou, China) and were used to generate seedlings for culturing the *M. incognita*. For the nematodes culture, one-month-old tomato seedlings were transplanted into pots (7 × 7 × 8 cm) with second stage juveniles of root-knot nematode-infected peat moss (Gui Yu Xin Nong Technology Co., Ltd., Nanning, China) and maintained at 25 ◦C with a 14 h light (22000 Lux) and 10 h dark photoperiod treatment within a GXZ-280C incubator (Jiangnan Instrument Factory, Ningbo, China). Tomato roots were collected 35 days after inoculation and were gently rinsed with tap water. Eggs were then extracted with 1% NaOCl [25] and hatched at 25 ◦C using the modified Baermann funnel method [26]. Eggs were put in 30 ìm pore sieves, nested in petri dishes (6 cm-diameter) containing 3 mL distilled water, and the fresh J2s in water were then collected on the day of experiment and used for subsequent experimentation.

#### *2.2. Fermentation Filtrate Preparation*

*A. japonicus* ZW1 from soil was deposited in the China Center for Type Culture Collection (accession number CCTCC No. M 2014641) and GenBank (accession number KR708636.1). One cm2 potato dextrose agar (PDA) with a fresh culture of *A. japonicus* ZW1 (cultured 3-5 days at 25 ◦C) was inoculated in triangular flasks with 100 mL Czapek medium (NaNO3 0.2 g, KCl 0.05 g, FeSO4 0.001 g, K2HPO4 0.1 g, MgSO4 0.05 g, Sucrose 3.0 g, H2O 100 mL) and incubated in a MQD-S2R shaker (Minquan Instrument Co., Ltd., Shanghai, China) at 150 rpm and 25 ◦C [27] for 3 consecutive weeks, with 10 triangular flasks replicates per week. Czapek medium without inoculation was used as a

negative control. At the end of the 3-week period, fermentation broth from a total of 30 conical flasks was then filtered using 0.45 μm Millipore filters (Whatman, Clifton, NJ, USA) and 1-week fermentation filtrate (1-WF), 2-WF, and 3-week fermentation filtrate (3-WF) were prepared. The concentration of 2.5% (i.e., fermentation filtrate volume: sterilized water volume = 1:39), 5% (1:19), 10% (1:9), 20% (1:4) and 50% (1:1) of 1-week fermentation filtrate(1-WF), 2-week fermentation filtrate (2-WF), and 3-week fermentation filtrate (3-WF) were used and 20% Czapek medium and sterilized water were used as control.

### *2.3. E*ff*ect of Fermentation Filtrate on Meloidogyne Incognita Egg Hatching*

Fresh eggs were treated with 2.5%, 5%, 10%, 20%, and 50% 1-WF, 2-WF, and 3-WF; and also 20% Czapek medium and sterilized water as controls. The specific experimental conditions were as follows: approximately 100 eggs and 200 μL of different concentrations of fermentation filtrate were dispensed into each well of 96-well plate, with 4 replicates for each treatment. Additionally, all experiments were performed in triplicate. The initial number of eggs was counted, and the hatched J2s were recorded using an inverted microscope (Ti-S, Nikon Instruments Inc., Tokyo, Japan) at 0, 3, 6, 9, 12, 15 d after exposure in the dark at 25 ◦C. The cumulative hatching rate was calculated using the following formula: cumulative hatching rate = (the number of hatched J2s)/(the initial number of eggs) × 100%.

#### *2.4. Nematicidal Activity of Fermentation Filtrate on Meloidogyne Incognita J2s*

Approximately 60 fresh J2s were contained in each well of a 96-well plate and treated with 200 μL of 2.5%, 5%, 10%, 20%, and 50% 1-WF, 2-WF, and 3-WF, 20% Czapek medium and sterilized water. The number of dead nematodes were counted using a Ti-S Nikon microscope (Nikon Instruments Inc., Tokyo, Japan) at 6, 12, 24, 48 h after treatment with the solutions and pictures were taken at each time point except for 48 h. It wax determined whether he bodies of dead J2s were straight and lacking movement even after mechanical prodding [28,29]. The test was conducted at 25 ◦C in the dark and the experiment was replicated 4 times. J2 mortality was calculated for each well as follows: mortality = (the number of dead J2s/total J2s) × 100%. This experiment was performed a total of three times.

#### *2.5. Scanning Electron Microscopy Observations*

J2s were treated with 10% 2-WF for 10 h and subsequently analyzed with scanning electron microscopy (SEM) using the approach as described below [30,31]. In preparation for the microscopic evaluations, J2 specimens were fixed in 2.5% glutaraldehyde with 0.1 M phosphate buffer (pH 7.2) at 4 ◦C overnight and subsequently washed 3 times in 0.1 M phosphate buffer. Afterwards, they were then fixed in 1% osmium tetroxide for 2 h, washed 3 times in 0.1 M phosphate buffer again, dehydrated in a graded series of ethanol, critical point dried with Quorum K850 critical dryers (Emitech, East Sussex, England, UK) and finally sputter coated with MSP-2S gold-palladium (IXRF, Austin, TX, USA). Prepared J2 specimens were observed using a SU8100 scanning electron microscope (Hitachi, Tokyo, Japan) operating at 3.0 kV accelerating voltage.

#### *2.6. Transmission Electron Microscopy Observations*

The technical approach was very similar to the aforementioned method described for 'scanning electron microscopy observations'; however, after J2s were dehydrated with ethanol, they were subsequently embedded in Araldite (Sigma-Aldrich, Sigma-Aldrich LLC., Darmstadt, Germany). To enable evaluation of the specimens, ultrathin sections (70 nm) were obtained using an EM UC7 ultramicrotome (Leica, Wetzlar, Germany) with a Diatome Ultra 45◦ diamond knife (Diatome Ltd., Helmstrasse Nidau, Switzerland). Sectioned samples were then stained with uranyl acetate and lead citrate using carbon film copper 500 mesh [30,32]. Sections of the J2 bodies were then observed using an HT7700 transmission electron microscope (Hitachi, Tokyo, Japan) operating at an 80.0 kV accelerating voltage.

#### *2.7. Greenhouse Experiment*

Thirty day old (3–4 leaf stage) healthy tomato seedlings (cv. Xin Bite 2 F1) were transplanted in a pot (785 cm3) containing 250 g autoclaved and dried peat moss. A total of 2000 fresh J2s were inoculated in each pot at 3 days after transplanting. Subsequently, 130 mL of 20% and 50% 2-WF were used in this experiment and applied in pots. 20% of Czapek medium and tap water were utilized as controls. A randomized design with 6 replicates for each treatment group was used for the pot experiment and all materials were maintained after inoculation at 25 ◦C in a greenhouse with a 14 h light and 10 h dark photoperiod. Thirty-five days after transplantation, tomato roots were collected and gently washed with tap water to remove residual materials. Plant height, root fresh weight, and the total number of galls and eggs per plant root system were determined. The eggs were extracted separately from plants with a 1% NaOCl method as previously described [25] and were subsequently collected in beakers with water. Afterwards, 50 μL of a well-mixed egg suspension solution were transferred to a counting dish to enable egg count determination. Eggs were counted three times and the total number of eggs in the entire suspension was calculated. This experiment was repeated twice.

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

Two-hundred mL of fresh 2-WF was dispensed into two 100 mL beakers respectively. One of the beakers was boiled in a microwave oven at 100 ◦C, whereas the second beaker was maintained at room temperature. The fermentation filtrate from two beakers were diluted to 10% and sterilized water was used as a control. Nematicidal activity was then conducted as described above and the experiment was triplicated.

For the analysis of storage time, the experiment was set up for 1-, 2-, and 3-week old 2-WF at 4 ◦C and 25 ◦C in dark, respectively; with 4 replicates for each treatment. After storage, the 2-WF solution was filtered through a sterile 0.45 μm polyethersulfone filter (Whatman, Clifton, NJ, USA) and subsequently diluted to a 10% solution in sterilized water. Sterilized water alone was used as a negative control. The nematicidal activity was measured as described above and this experiment was repeated 3 times.

#### *2.9. Evaluation of the Strain Fermentation Filtrate on The Germination of Crop Seeds*

In this study, the effect of 2-WF of *A. japonicus* ZW1 was evaluated on seed germination of various crops, e.g., from corn (Qingnong 13), wheat (Mianmai 41), cowpeas (Shanlv), cabbage (Green column), cucumbers (Liaoning 8), rice (Teyou 09103), tomatoes (Hongyingguo 808), and soybeans (Ludou 4). First, healthy seeds were surface sterilized with 2% NaOCl for 3 min and subsequently rinsed 5 times with sterilized water [33]. Seeds were treated with 10% and 20% 2-WF in triplicates across 3 independent experiments, with sterilized water used as a negative control. The sterilized crop seeds were then exposed to the fermentation filtrate in a moist chamber and incubated for several days in the dark at room temperature (25 ◦C). Sprouted seeds were counted every day until the seed germination rate no longer changed. The seed germination rate was calculated as: (number of germinated seed/total tested seeds) × 100%.

#### *2.10. Isolation and Structural Determination of Aspergillus Japonicus ZW-1 Nematicidal Metabolites*

Eight litre of *A. japonicus* ZW-1 2-week fermentation broth was filtered through 8 layers of muslin gauze, then concentrated to 500 mL using rotary evaporation (Hei-VAP Core ML G3, Instruments GmbH & Co. Heidolph, KG, Schwabach, Germany) at 55 ◦C. The crude extract (15.6 g) from *A. japonicus* ZW-1 fermentation broth was extracted with 1-butanol and evaporated at 40 ◦C until dry, dissolved in methanol (MeOH) and chromatographed on methylated sephadex LH20 (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) using MeOH as eluent to give two fractions, the two fractions were dissolved in distilled water to make 2.0 mg mL−<sup>1</sup> aqueous solution for activity assay. One fraction showed activity against J2. This active fraction was dissolved in the chloroform, at which point white

crystals formed. The solution was filtered through cotton which was then washed 20 times using chloroform and dried at room temperature to get the purified active compound.

The chemical structures of the active compound were determined using nuclear magnetic resonance (NMR) analysis and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) analysis. 1H nuclear magnetic resonance (NMR) and 13C NMR spectra were acquired in MeOH with a Bruker AVANCE III HD600 spectrometer (Bruker Corporation, Faellanden, Switzerland) at 600 MHz for 1H NMR spectra and 125 MHz for 13C NMR spectra using tetramethylsilane as the internal standard. HR-ESI-MS analysis was performed using a Waters E2695 model ion trap mass spectrometer (Waters, Milford, MA, USA) [34]. The nematicidal activity of active compounds at different concentrations (1.25, 1.00, 0.75, 0.50, 0.25 mg mL<sup>−</sup>1) was measured as described above and this experimental approach was repeated 3 times. Sterilized water was used as a control.
