Soil samples were obtained from a field of Globodera sp. infected potato fields located in Weining County, Guizhou, China. In each field, 10 plots of a 5 × 5 m grid were chosen around infected potato plants, and an approximate volume of 250 mL of soil from the rhizosphere zone was collected in each grid (0–20 cm depth). To make a single composite sample, the separate samples from each plot were collected and combined in a bucket (Southey 1974).

To ensure homogeneity, the composite samples were thoroughly mixed. Cysts of Globodera sp. were extracted from a subsample of 500 mL soil that had been air-dried at 37°C for two days (Been and Schomaker 2000, Reid and Pickup 2005, Nurjanah et al. 2016). Using the Baunacke method (Baunacke 1922, Hallman and Viaene 2013), cysts were obtained from a 100 g subsample of dried soil by decanting floating cysts in water and collecting them on a 250 mesh sieve.

Subsequently, the cysts were surface sterilized for three minutes using 0.2% H₂O₂, followed by three rinses with distilled water. Each sterilized cyst was individually placed on a 1% water agar (WA) plate. The plates were incubated at room temperature and monitored regularly. Mycelia emerging from the cultured cysts were transferred to new potato dextrose agar (PDA) plates multiple times. N. stercicola strains exhibiting high nematicidal activity were preserved in the Culture Collection of the Department of Plant Pathology, Agriculture College, Guizhou University, under strain numbers GUCC2232 and GUCC2212.

To prepare potato dextrose broth (PDB) medium, 200 g potatoes were boiled in 1 L of distilled water for 30 minutes. Dextrose was then added to the filtrate after the mixture was filtered through double gauze. Afterward, 100 mL aliquots of the prepared PDB medium were placed into 250 mL conical flasks and were autoclaved for 30 minutes at 121°C. With a sterilized cutting blade, the single pure culture was cut into small pieces approximately 5 mm in diameter. Five pieces were added in 100 mL of sterilized PDB medium, which was then shaken at 28°C for 7 d at 200 rpm on a rotary shaker. Finally, the medium was filtered and stored at 4°C.

The fungal strains GUCC2212 and GUCC2232 were inoculated on PDA and cultivated for 7 d at 28°C. Colony morphology was documented using a stereomicroscope (Keyence VHX-7000 digital microscope). A compound light microscope (Zeiss Scope 5) equipped with an AxioCam 208 color camera was used to photograph conidiophores and conidia. To determine the mean size, 30 conidiogenous cells, 50 macroconidia, and 50 chlamydospores from each strains were mounted and measured randomly.

The fungal strains GUCC2212 and GUCC2232 were cultured on PDA for 7 d at 25°C. Using a sterile scalpel, the mycelia were carefully scraped from the plate's surface. Total genomic fungal DNA was extracted with a BIOMIGA Fungus Genomic DNA Extraction Kit (GD2416, BIOMIGA, San Diego, California, USA) following the manufacturer's instructions. The 5.8S nuclear ribosomal RNA gene was amplified and sequenced, together with the two flanking internal transcribed spacer (ITS), translation elongation factor (tef1) and large subunit (LSU) sections. The primer pairs and PCR amplification protocols ITS5/ITS4 (White et al. 1990a), EF1/EF2 (O'Donnell et al. 1998) and LR0R/LR5 (Perera et al. 2020) were used to PCR-amplify ITS, tef1 and LSU, respectively.

PCR amplifications were conducted in a reaction mixture containing 12.5 µL 2x Bench Top Taq Master Mix (Biomiga, AT1201, China), 1 µL each of 10 µM primers and 1 µL DNA template, with the final volume adjusted to 25 µL using distilled deionized water. PCR products were visualized on a 1% agarose gel through electrophoresis. Bidirectional sequencing was performed by Sangon Biotech Company (Chengdu, China).

Using BioEdit v.7.2.5 (Hill 1999), forward and reverse primer sequences for each gene were assembled and consensus sequences were merged with relevant sequences obtained from the National Center for Biotechnology Information (NCBI). Table 1 presents the sequences of the two strains investigated in this study, along with 11 reference strains acquired from GenBank (https://www.ncbi.nlm.nih.gov/genbank). Sequences for each locus were aligned using MAFFT v. 7.187 (Katoh and Standley 2013) and alignments were manually adjusted as necessary.

Utilizing the maximum likelihood (ML) approach Bayesian inference (BI) via the CIPRES web platform, a phylogenetic tree was created using the ITS, tef1 and LSU sequences as a concatenated dataset (Miller et al. 2010). Every 100 generations, trees were sampled and runs were automatically ended when the average standard deviation of split frequencies fell below 0.01. After removing the first 25% of samples, a 50% majority rule consensus tree was created. FigTree v1.4.3 (Rambaut and Drummond 2012) and Adobe Illustrator CC 2019 were used to visualize the generated tree.

To evaluate their pathogenicity against Globodera sp. eggs, pure fungal cultures were grown on PDA for 7–10 d. Conidial suspensions were prepared by flooding the PDA plates with double-distilled water (DDW) and the surface scraped. The suspension was diluted with DDW to achieve 1.0 x 10⁶ spores/mL using a haemacytometer.

For the egg parasitic ability test, eggs released from cysts by crushing them with a glass rod were suspended in DDW at a concentration of about 100 eggs per 100 µL suspension that was dropped into each well of a 24-multiwell plate containing each spore suspension with three replications. They were all incubated at 25°C and the number of eggs that were parasitized by fungi was examined every three days after incubation.

A 100 μL suspension of nematodes containing approximately 100 nematodes was placed into wells of a 96-well culture plate containing different concentrations (100%, 20%, 10%) of the fermentation broth. The distilled water was added into the control wells. The plate was incubated for 72 h at 28°C. After 12, 24, 48 and 72 h, nematodes were examined under a microscope. The nematodes were cleansed and put into distilled water at each timepoint to assess their motility as a sign of nematicidal activity. When nematodes remained immotile after being probed with a fine hair needle, they were judged dead and percentage mortality was calculated. For each concentration and the control, four replicates were examined.

A 5 mm-diameter disc of mycelium was removed from the margins of the fungal isolates, placed to the center of a petri dish containing 1% water agar (WA) and cultured for two weeks in the dark at 25°C. Petri dishes were infected with a 1 mL nematode slurry containing 1000 nematodes after the incubation period. The nematode suspension was separated into 4-5 drops and evenly distributed across the fungal colonies periphery. Control plates were made without fungus. Each strain was tested in four replicates. A compound light microscope (Zeiss Scope 5) with an AxioCam 208 color camera was used to acquire microscopic images.

The data were subjected to two-way analysis of variance (ANOVA), with concentration and post-treatment time (exposure period) serving as the main treatment effects and concentration x time as the interaction. Significant differences between means were determined at P<0.05 using Duncan’s multiple range. All statistical analyses were performed using MS Excel and SPSS statistics software (version 19.0). Figures were generated using Origin 2018.

The sequences of the PCR products obtained from strain GUCC2232 and GUCC2212 were uploaded to GenBank and subjected to Basic Local Alignment Search tool (BLAST) analysis. In the phylogenetic tree (Fig. 1), GUCC2232 and GUCC2212 clustered with the type culture of N. stercicola (CBS 260.54). The ex-type cultures of species, F. stercicola and F. witzenhausenense, also cluster within this clade. Furthermore, the lack of clear distinctive morphological traits between these species, coupled with evidence of significant recombination within this clade according to the PHI test (Šišić et al. 2018), supports their synonymy as N. stercicola.

Figure 1.  

Phylogenetic tree generated from RAxML and Bayesian inference (BI) analysis based on combined ITS, tef1 and LSU sequence data of N. stercicola (GUCC2232, GUCC2212). N. tonkinensis was designated as the outlier taxon. The scale bar indicates 0.003 nucleotide changes per site.

The colonies of strain GUCC2232 on PDA after 7 d of incubation at 28°C exhibit a white to yellowish-white surface, with abundant cottony aerial mycelium throughout the colony. The reverse pigmentation is yellow at the center and fades to yellowish-white at the margin. The colony margin is undulate with an entire edge(Fig. 2a, b). The odour is strong moldy. Scant production of erect conidiophores, tapering uniformly from base to tip from the agar surface and aerial mycelium. Tip of the phialid with periclinical thickening, collared at most slightly flared (Fig. 2c, d, e). Conidia often held in clear, colourless drops of liquid, 0, 1, 2, 3 (- 4) septate: zero-septate ellipsoidal 11.40 × 4.12 μm; one-septate 19.27 × 5.25 μm; two septate 25.15 × 5.22 μm; three septate slightly curved, apical cell rounded and undeveloped foot cell 28.38 × 5.93 μm; 4 septate (n = 1) slightly curved with a slightly hooked apical cell and barely notched basal cell 27.29 × 6.13 μm (Fig. 2j-m). Chlamydospores formed abundantly after 7 d, unicellular, ovoid to ellipsoidal, single or in chains, terminal on hyphae or intercalary, consisting of enlarged, thick-walled vegetative cells: unicellular 6.29-6.84 μm; multicellular 10.85-11.57 μm in diam (Fig. 2f-i).

Figure 2.  

Colony and microscopic features of N. stercicola (GUCC2232). a-b Top and bottom images of GUCC2232 cultured on PDA c Monophialides and microconidia d-e short Monophialides with false head f-i Chlamydospores j-m multiseptate macroconidia. Scale bars: c-g, i = 50 μm; h, j-m = 10 μm.

The colonies of the GUCC2212 strain on PDA after 7 d of incubation at 28°C exhibited a brownish-grey center and yellowish-white margin, with radial V-shaped stripes composed of dense white flat mycelium stretching from the colony center to the edge. The aerial mycelium was scarce and the colony was fine with abundant production of cream slimy sporodochia at the margins. The reverse pigmentation was reddish-grey in the center, fading to yellowish white at the margin with irregularly distributed brownish-grey pigmented areas. The colony margin was entire to undulate (Fig. 3). The odour is sweet to moldy. The strain exhibited abundant production of erect, mononymous conidiophores from the agar surface and aerial mycelium. Tip of the Conidia often held in clear, colourless drops of liquid 0, 1 and 3 septate, with the zero-septate ellipsoidal or having a rounded apex and truncate base measuring 11.51 × 3.39 μm; one-septate ellipsoidal measuring 20.38 × 4.39 μm; and three-septate slightly curved or arcuate with a rounded apical cell and poorly developed basal cell measuring 28.05 × 5.95 μm (Fig. 3h-k).

Figure 3.  

Colony and microscopic features of N. stercicola (GUCC2212). a-b Top and bottom images of GUCC2212 cultured on PDA c Monophialides and microconidia d-g Chlamydospores h-k short Monophialides multiseptate macroconidia. Scale bars: c-g = 50 μm; i-k = 10 μm.

Although the observed morphological characteristics of GUCC2232 and GUCC2212 were not entirely consistent with those described for F. stercicola and F. witzenhausenense by Šišić et al.(Šišić et al. 2018), we conclude that these strain belong to the N. stercicola species based on a combination of morphological observations and phylogenetic analysis.

The fungal strains GUCC2212 and GUCC2232 were capable of infecting the eggs of Globodera sp. in vitro. Two strains infected and destroyed the Globodera sp. eggs (Fig. 4). At the same temperature with the same concentration, the parasitic rates differed at different times. The parasitic rate showed an overall upward trend with the extension of time and the parasitic rate of nematode eggs reached its highest 9 d after treatment. At this time, the conidia of N. stercicola were adsorbed or colonized on the shell surface of the nematode eggs and the hyphae surrounded the eggs and penetrated the eggshell (Fig. 4d-i), while no fungal growed from the control eggs (Fig. 4a-c). After 9 d of treatment with the same concentration of conidial suspension, the parasitical ratio of GUCC2212 was 21.19% and GUCC2232 was 22.41%, respectively. The relative parasitic rates of the two strains were very similar (Table 1).

Figure 4.  

Globodera sp. eggs parasitized by the strains of N. stercicola (GUCC2212, GUCC2232). a-c Healthy egg d Hypha contact egg shell e-i Hyphae pass egg shell. Scale bars = 50 μm. Data represent the mean±SE (n = 4).

In order to obtain more microorganism resources to control plant-parasitic nematodes (PPNs), control efficiency of two fungal strains N. stercicola (GUCC2212) and N. stercicola (GUCC2232) on three PPNs were evaluated. The nematicidal activity of the N. stercicola fermentation filtrate was significantly different from that of PDB medium alone. PDB did not exhibit nematicidal or nematicidal activity because nematode mortality in PDB was statistically similar to that in sterilized water (P<0.05).

The mortality rate in A. besseyi was 12%, 100%, 100% and 100% at 12, 24, 48 and 72 h, respectively (Fig. 5A). The mortality rate in B. xylophilus increased rapidly, at 4%, 67%, 96% and 97% at 12, 24, 48 and 72 h, respectively (Fig. 5B).

Figure 5.  

In vitro nematicidal activity of the fermentation filtrate of Neocosmospora stercicola (GUCC2212 and GUCC2232) against three species of plant pathogenic nematodes: A A. besseyi B B. xylophilus C D. destructor. *Note: Different letters indicate a significant difference between the different treatments within a given time point. Lowercase letters indicate significantly different means at P<0.05. Data represent the mean±SE (n = 4).

Meanwhile, the nematicidal activity of the undiluted fermentation filtrate of N. stercicola (GUCC2212) was the highest nematicidal activity against D. destructor, with mortality rates of 47%, 90%, 96% and 100% at 12, 24, 48 and 72 h, respectively (Fig. 5C). Although the corrected mortality rate of nematodes after fermentation broth treatment should gradually decrease with increasing dilution factor, 10% preparation of the fermentation broth also exhibited a significant level of nematicidal activity at 72 h, relative to the controls against A. besseyi, B. xylophilus and D. destructor (P<0.05) (Fig. 5).

Although the two strains differed in their nematicidal activity against the three PPNs, collectively the data indicate that the fermentation broth of N. stercicola strains exhibited different levels of nematicidal activity against three different species of nematodes. Current investigations suggested that fermentation of N. stercicola could be used to control PPNs.

Nematodes moved freely on 1%WA plates of GUCC2212/GUCC2232 cultures during the initial 12 h of coincubation. However, their movement became increasingly limited between 12 and 24 h due to entanglement with hyphae. By 24-48 h of coincubation, nematodes were completely entrapped and unable to move. Two days later, the hyphae began to attach to the surface of entrapped nematodes, completely engulfing them. Following 72 h of coincubation, the number of fungal colonies increased, the body wall of the entrapped nematodes dissolved and their internal contents were consumed, leaving only traces of the entrapped nematodes behind (Fig. 6a-c, Fig. 7a-c).

Figure 6.  

Predatory activity of N. stercicola (GUCC2212) against three plant pathogenic nematodes on water agar (WA). a-c Predatory activity against A. besseyi at 24, 48 and 72 h d-f Predatory activity against B. xylophilus at 24, 48 and 72 h g-i Predatory activity against D. destructor at 24, 48 and 72 h). Scale bars = 200 μm. Data represent the mean±SE (n = 4).

Figure 7.  

Predatory activity of N. stercicola (GUCC2232) against three plant pathogenic nematodes on water agar (WA). a-c Predatory activity against A. besseyi at 24, 48 and 72 h d-f Predatory activity against B. xylophilus at 24, 48 and 72 h g-i Predatory activity against D. destructor at 24, 48 and 72 h). Scale bars = 200 μm. Data represent the mean±SE (n = 4).

Under in vitro conditions, the two strains of N. stercicola were cultured on 1% WA and separately co-incubated with three different species of nematodes. The number of total nematodes entrapped in five independent microscopic fields of view was recorded over 72 h. The results indicated that N. stercicola (GUCC2212, GUCC2232) exhibited predatory activity against nematodes at different points in time. In general, the percentage of trapped nematodes increased with the time of coincubation for A. besseyi. The predation activity of N. stercicola (GUCC2212,GUCC2232) was greatest against A. besseyi, with an average percentage of 35.19% and 37.96% respectively at 72 h (P<0.05) (Table 2).