Novel ectophytic nematode antagonistic fungi and its potential application for nematode biocontrol

doi:10.21203/rs.3.rs-2234705/v1

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Novel ectophytic nematode antagonistic fungi and its potential application for nematode biocontrol

https://doi.org/10.21203/rs.3.rs-2234705/v1

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Dynamic of plant-parasitic nematodes population in soil is closely related to soil microorganisms. Fungi from Heterodera zeae cysts were isolated to explore the natural decline phenomenon of H. zeae population. A polyphasic method was used to identify the nematode-destroying fungal, including phylogenetic analysis of partial ITS, BenA, CaM, and RPB2 gene sequences, as well as macro- and micro-morphological analyses. The nematicidal activity of strain GX1 against H. zeae was assessed in vitro and greenhouse. Our findings revealed that strain GX1 is a novel species of Talaromyces, which named Talaromyces cystophila. It had strong parasitic and lethal effect on the H. zeae cysts with 91.11% parasitism on cysts at 3 d after treatment, and second-stage juveniles (J2s) and eggs inside the cysts appeared dense vacuoles and loss of vitality. The spore suspension and fermentation filtrate of T. cystophila inhibited significantly the hatching of H. zeae eggs and showed potential nematicidal activities on J2s. The hatching inhibition rates of eggs exposed to 1 × 10⁸ cfu/ml spore suspensions or 20% 1-week fermentation filtrate (1-WF) for 15 days were 98.56% and 100%, respectively. The mortality of J2s exposed to 1 × 10⁸ cfu/ml spore suspension reached 100% at 24 h, exposure to 50% 2-WF was 98.65% and 100% at 24 h and 48 h, respectively. Greenhouse experiments revealed that the spore suspension and fermentation broth considerably decreased H. zeae reproduction by 56.17%-78.76%. T. Cystophila is a potential biocontrol strain with parasitic and nematicidal activity, which deserves attention and application.

Heterodera zeae

Talaromyces cystophila

Biological control fungi

Nematicidal activity

Nematode-destroying

Maize (Zea mays L., cv. Azam.) is an essential cereal crop grown worldwide, providing a major resource for food, fodder, and industrial materials (Zhang et al. 2020). During the growth of maize, the yield of maize is frequently limited by various biotic and abiotic stress factors, such as drought, salinity, nutrient deficiencies, fungi, bacteria, insects and nematodes (Li et al. 2018). Heterodera zeae is considered one of the most important nematode pests of maize (Singh and Rathore 2001, Sikandar et al. 2021), and can cause 21–29% corn yield losses (Srivastava and Chawla 2005; Kumhar et al. 2018). Koshy et al. (1971) reported H. zeae from maize (Zea mays L.) in Rajasthan state, India. Subsequently, then recorded in other countries, including Pakistan, the United States, Thailand, Nepal, Portugal, Greece, and Afghanistan (Asghari et al. 2013). In 2016, H. zeae was reported on maize in Guangxi province, China. (Wu et al. 2017). As higher temperature (33℃) is conducive to hatching and reproduction of the nematode, with the global climate warming, it is a potential risk to maize production area, and control measures are needed to prevent any further spread.

For decades, the control of nematodes has relied heavily on chemical nematicides, but currently available chemical nematicides are being withdrawn because of their notorious toxicity to wildlife and human health (Li et al. 2015). Therefore, it is urgent to develop efficient and environmentally safe alternatives to chemical pesticides (Seong et al. 2021). Microorganisms have recently attracted much attention as potential nematode bio-control agents due to their nematicidal activities and environmental friendly nature (Lu et al. 2014). Paecilomyces lilacinus (Ganaie and Khan 2010), Trichoderma harzianum (Naserinasab et al. 2011), and Pochonia chlamydosporia (Muthulakshmi et al. 2012) are commercial products that are frequently used for the biocontrol of plant-parasitic nematodes. For example, P. lilacinus has been used as a biocontrol agent and widely used for plant parasitic nematode control in China, the United States, India, Germany, and Philippines. Among them, PL plus developed by BCP South Africa based on P. lilacinus as an active ingredient, was used to control pathogenic nematodes in important economic crops such as vegetables, tobacco, banana, and citrus (Li et al. 2015).

Talaromyces, kind of saprophytic fungi widely distributed in nature, are closely related to plants, animals, and humans. The species belonging to the genus Talaromyces, produce anticancer, antibacterial, and antifungal compounds (Nicoletti et al. 2018; Zhai et al. 2016), natural colorants (Frisvad et al. 2013) and enzymes (Antonopoulou et al. 2018; Pol et al. 2012). Several Talaromyces species have shown potential as biocontrol agents of nematodes (Ait Hamza et al. 2016; Farhat et al. 2022). Notably, Guo et al. (2012) isolated and characterized a novel class of potent nematicidal thermolides from the thermophilic fungus Talaromyces thermophilus. Thermolides 1 and 2 among the PKS-NRPS hybrid metabolites displayed potent inhibitory activity against three species nematodes (Meloidogyne incognita, Bursaphelenches siylopilus, Panagrellus redivevus) with LC₅₀ values of 0.5-1 µg/ml, as active as commercial avermectins. Undoubtedly, the genus Talaromyces is an excellent natural repository for novel compounds that can produce many biologically active substances (Niu et al. 2014; Yilmaz et al. 2014). Besides, some species have been proven to be effective biological control agents for soil-borne diseases (Sun et al. 2020b). Talaromyces pinophilus shows antagonistic activity and parasitic effects against Rhizoctonia solani and Botrytis cinerea (Alagesaboopathi 1994; Abdel-Rahim and Abo-Elyousr 2018). It releases cellulase, which can hydrolyze plant lignocellulose (Belancic et al. 1995) and increase nutrient uptake (N, P, and K) (Maity et al. 2014). Furthermore, it is essential for producing soil humus and stimulating plant development.

Therefore, a strain with a strong ability to parasitize nematodes and nematicidal activity isolated from the H. zeae cyst was evaluated systematically, to explore the mechanism of natural decline of H. zeae population in the soil. Our main objectives were to (i) isolate and characterize strain GX1 based on morphological and molecular (ITS, BenA, CaM and RPB2 gene sequences) characteristics; (ii) evaluate strain GX1's parasitism and nematicidal activity via in vitro assay and greenhouse pot experiment.

Sample collection and strain isolation

Cysts were collected in Xiaopingyang Town, Xingbin District, Laibin City, Guangxi, China (109°07′45′′E, 23°24′13′′N) from maize fields exhibiting natural declines of H. zeae populations. The zigzag sampling method was used to obtain 50 soil cores with a diameter of 2.5 cm and a depth of 20 cm. The H. zeae cysts were extracted from soil by the modified sucrose-flotation and centrifugation technique (Mo et al. 2021). Cysts were surface disinfection with 0.5% NaOCl for 3 min and rinsed with sterile water. The disinfected cysts were placed in water agar Petri dishes containing streptomycin and chlortetracycline (SC). The dishes were incubated at 25°C, and fungal growth was examined at 1–2 day intervals up to 2 weeks of incubation. Fungal mycelia growing from cysts were first transferred to Potato dextrose agar (PDA, Difco, Detroit, MI) plates (Chen and Chen 2012). The isolates were purified and tested its infectivity on cysts, the parasitism rate of strain GX1 was over 90%. In this study, the strain GX1 was identified and deposited in the China General Microbiological Culture Collection (CGMCC) as CGMCC40002. Subsequently, a series of nematicidal trials of this strain were carried out.

Morphology of strain GX1

Strains were characterized under standardized conditions using media and methods described by Samson et al. (2010). Culture media used for characterization include Czapek yeast autolysate agar (CYA), malt extract agar (MEA, Oxoid), oatmeal agar (OA), and yeast extract sucrose agar (YES). Inoculations were prepared from spore suspensions in a semi-solid agar solution containing 0.2% agar and 0.05% Tween80 (Pitt 1979). Then, inoculated at three points on 90 mm Petri dishes and incubated for 7 d at 25°C in darkness. Additional CYA plates were incubated at 30 and 37°C, while additional MEA plates were incubated at 30°C. After 7 d of incubation, colony diameters were recorded. The colony texture, degree of sporulation, obverse and reverse colony colors, the production of soluble pigments and exudates were noted (Sun et al. 2020a).

Microscope preparations were made from 7 d old colonies grown on MEA. Lactic acid (60%) was used as mounting fluid, and 96% ethanol was applied to remove the excess conidia. Microscopic observation and photographs were performed with a microscope (Axio Imager, Z2, Carl Zeiss).

DNA extraction, PCR amplification and sequencing

DNA was extracted from cultures grown on MEA for 7 d using a Rapid Fungal Genomic DNA Isolation Kit (Sangon Biotech, Shanghai, China). Polymerase chain reaction (PCR) amplification of the Internal Transcribed Spacer (ITS), β-tubulin (BenA), Calmodulin (CaM), and RNA polymerase II second largest subunit (RPB2) gene regions and sequencing reactions followed previously described methods (Table 1). The PCR was carried out in a 20 µL reaction mixture containing 0.5 µL of each primer (10 pM/µL), 1.0 µL genomic DNA (10 ng/µL), 10 µL 2 × PCR MasterMix buffer (0.05 U/µL Taq polymerase, 4 mM MgCl₂, 0.4 mM dNTPs) and 8 µL double-distilled water (Wei et al. 2021). PCR products were examined by electrophoresis in a 0.8% (w/v) agarose gel in 1 × TAE buffer (0.4 M Tris, 50 mM NaOAc, 10 mM EDTA, pH 7.8) with 100 bp DNA ladder at 120V for 20 min. The gels were stained with ethidium bromide (0.5 µg/ml) for 15 minutes and then detected using the Syngene G: Box camera (Synoptics, Cambridge, UK). The purified PCR products were sequenced directly with the above primer pairs using an ABI 3730-XL DNA sequencer.

Table 1

Primer sequences and reaction conditions for PCR amplification
Locus	Primers	Direction		References	Reaction cycle conditions
internal transcribed spacer (ITS)	ITS1	Forward	TCC GTA GGT GAA CCT GCG G	White et al. (1990)	94°C, 5 min, 94°C, 45 s, 55°C, 45 s, 72°C, 1 min, (35 cycles) 72°C for 7 min
internal transcribed spacer (ITS)	ITS4	Reverse	TCC TCC GCT TAT TGA TAT GC	White et al. (1990)
calmodulin (CaM)	CMD5	Forward	CCG AGT ACA AGG ARG CCT TC	Hong et al. (2006)	94°C, 5 min, 94°C, 45 s, 55°C, 45 s, 72°C, 1 min, (35 cycles) 72°C for 7 min
calmodulin (CaM)	CMD6	Reverse	CCG ATR GAG GTC ATR ACG TGG	Hong et al. (2006)
β-tubulin (BenA)	Βt2a	Forward	GGT AAC CAA ATC GGT GCT GCT TTC	Glass and Donaldson (1995)	94°C, 5 min, 94°C, 30 s, 55°C, 45 s, 72°C, 1 min, (35 cycles) 72°C, 7 min
β-tubulin (BenA)	Bt2b	Reverse	ACC CTC AGT GTA GTG ACC CTT GGC	Glass and Donaldson (1995)
RNA polymerase II second largest subunit (RPB2)	5F	Forward	GAY GAY MGW GAT CAY TTY GG	Liu et al. (1999)	94°C, 5 min, 94°C, 45 s, 60°C, 45 s, 72°C, 1 min, (35 cycles) 72°C, 7 min
RNA polymerase II second largest subunit (RPB2)	7CR	Reverse	CCC ATR GCT TGY TTR CCC AT	Liu et al. (1999)

Phylogenetic analysis

For the taxonomy of Talaromyces, a four-gene phylogeny combining ITS, BenA, CaM and RPB2 sequences was used. Raw sequences were proofread and edited manually with BioEdit 7.0.9 (Hall 1999). Sequences of strain GX1 generated in this study were deposited and made available in GenBank. The newly generated sequences were included in a dataset including sequences obtained from Yilmaz et al. (2014) and supplemented with sequences of new species that were subsequently described (Table 2) (Sun et al. 2020a; Visagie et al. 2015). The dataset for each gene was aligned using the MUSCLE software included in the MEGA v. 7.0.26 software package (Tamura et al. 2013). The aligned sequences were trimmed to generate sequence matrices. The aligned ITS-BenA-CaM-RPB2 data were analyzed separately using Maximum Likelihood (ML) and Bayesian Inference (BI). The ML analyses were performed in MEGA v. 7.0.26 (Tamura et al. 2013), and the best model was selected based on the lowest Bayesian Information Criterion (BIC). ML analysis was done by calculating an initial tree using BioNeighbour-Joining (BioNJ) and the subsequent Heuristic search done with the Nearest-Neigh-bour-Interchange (NNI) option (Yilmaz et al. 2016), and bootstrap support was calculated using 1000 replicates. Bayesian (BI) analyses were performed on MrBayes v. 3.2.2 (Ronquist et al. 2012) based on the models selected by the MrModelTest 2.31 (Nylander 2004). The analysis was run until the standard deviation of split frequencies reached less than 0.01, indicating convergence had been reached. The sample frequency was set at 100 and the first 25% of trees were removed as burn-in. The resulting trees were visualized with FigTree v1.4.4. Bayesian inference (BI) posterior probabilities (pp) values and bootstrap (bs) values were labeled on nodes. Values less than 0.95 pp and 75% bs were not showed.

Table 2

The 65 species of *Talaromyces* and their four genetic markers used for molecular phylogenetic analysis in this study
Species	Strains	Genetic markers
Species	Strains	ITS	BenA	CaM	RPB2
Talaromyces aerius	CBS 140611 T = CGMCC3.18197 = DTO 317-C7	KU866647	KU866835	KU866731	KU866991
T. aerugineus	CBS 350.66 T = BDUN 276 = IMI 105412	AY753346	KJ865736	KJ885285	JN121502
T. aculeatus	CBS 289.48 T = ATCC 10409 = IMI 040588 = NRRL 2129 = NRRL A-1474	KF741995	KF741929	KF741975	MH793099
T. adpressus	CBS 140620 T = CGMCC3.18211 = DTO 317-G4	KU866657	KU866844	KU866741	KU867001
T. albisclerotius	CBS 141839 T	MN864276	MN863345	MN863322	MN863334
T. albobiverticillius	CBS 133440 T = DTO 166-E5 = YMJ 1292	HQ605705	KF114778	KJ885258	KM023310
T. allahabadensis	CBS 453.93 T = ATCC 15067 = CBS 304.63	KF984873	KF984614	KF984768	KF985006
T. angelicae	KACC 46611 T	KF183638	KF183640	KJ885259	KX961275
T. annesophieae	CBS 142939 T = DTO 377-F3 = JW9011	MF574592	MF590098	MF590104	MN969199
T. apiculatus	CBS 312.59 T = ATCC 18315 = FRR 635 = IMI 068239	JN899375	KF741916	KF741950	KM023287
T. assiutensis	CBS 147.78 T	JN899323	KJ865720	KJ885260	KM023305
T. atroroseus	CBS 133442 T = IBT 32470 = DTO 178-A4	KF114747	KF114789	KJ775418	KM023288
T. austrocalifornicus	CBS 644.95 T = IBT 17522	JN899357	KJ865732	KJ885261	MN969147
T. bohemicus	CBS 545.86 T = CCF 2330 = IAM 14789	JN899400	KJ865719	KJ885286	JN121532
T. boninensis	CBS 650.95 T = IBT 17516	JN899356	KJ865721	KJ885263	KM023276
T. borbonicus	CBS 141340 T = DTO 351-D3	MG827091	MG855687	MG855688	MG855689
T. brasiliensis	CBS 142493 T = URM 7618	MF278323	LT855560	LT855563	MN969198
T. brunneus	CBS 227.60 T = ATCC 18229 = FRR 646 = IFO 6438 = IHEM 3907 = IMI 078259 = MUCL 31318	JN899365	KJ865722	KJ885264	KM023272
T. cecidicola	CBS 101419 T = DAOM233329	LT559084	FJ753295	KJ885287	KM023309
T. chloroloma	DAOM 241016 T = CV 2802	FJ160273	GU385736	KJ885265	KM023304
T. cinnabarinus	CBS 267.72 T = ATCC 26215 = NHL 2673	JN899376	AY753377	KJ885256	JN121477
T. cnidii	KACC 46617 T	KF183639	KF183641	KJ885266	KM023299
T. coalescens	CBS 103.83 T	JN899366	JX091390	KJ885267	KM023277
T. columbinus	NRRL 58811 T	KJ865739	KF196843	KJ885288	KM023270
T. convolutus	CBS 100537 T = IBT 14989	JN899330	KF114773	MN969316	JN121414
T. dendriticus	CBS 660.80 T = IMI 216897	JN899339	JX091391	KF741965	KM023286
T. diversiformis	CBS 141931 T = CGMCC 3.18204 = DTO 317-E3	KX961215	KX961216	KX961259	KX961274
T. diversus	CBS 320.48 T = ATCC 10437 = DSM 2212 = IMI 040579 = IMI 040579ii = NRRL 2121	KJ865740	KJ865723	KJ885268	KM023285
T. erythromellis	CBS 644.80 T = FRR 1868 = IMI 216899	JN899383	HQ156945	KJ885270	KM023290
T. fuscoviridis	CBS 193.69 T = DTO 258-I7 = IBT 14846 = IBT 32646	KF741979	KF741912	KF741942	MN969156
T. heiheensis	CGMCC 3.18012 T	KX447526	KX447525	KX447532	KX447529
T. helicus	CBS 335.48 T = ATCC 10451 = DSM 3705 = IMI 040593 = NRRL 2106	JN899359	KJ865725	KJ885289	KM023273
T. islandicus	CBS 338.48 T = ATCC 10127 = IMI 040042 = MUCL 31324 = NRRL 1036	KF984885	KF984655	KF984780	KF985018
T. lentulus	AS3.15689 T = NN071323	KY007088	KY007104	KY007096	KY112586
T. liani	CBS 225.66 T = ATCC 18325 = ATCC 18331 = IMI 098480 = NRRL 3380 = VKM F-301	JN899395	JX091380	KJ885257	KX961277
T. loliensis	CBS 643.80 T = ATCC 52252 = FRR 1798 = IMI 216901 = MUCL 31325	KF984888	KF984658	KF984783	KF985021
T. mae	AS3.15690 T = NN071328	KY007090	KY007106	KY007098	KY112588
T. minioluteus	CBS 642.68 T = DTO 304-C4 = IMI 089377 = MUCL 28666	JN899346	MN969409	KJ885273	JF417443
T. minnesotensis	CBS 142381 T = FMR 14265 = UTHSC DI16-144	LT558966	LT559083	LT795604	LT795605
T. musae	CBS 142504 T = DTO 366-C5	MF072316	MF093729	MF093728	MF093727
T. piceus	CBS 361.48 T = ATCC 10519 = IMI 040038 = NRRL 1051	KF984792	KF984668	KF984680	KF984899
T. pigmentosus	URM 7624 T = CBS 142805	MF278330	LT855562	LT855565	LT855568
T. pinophilus	CBS 631.66 T = ATCC 36839 = CECT 2809 = DSM 1944 = IAM 7013 = IMI 114933	JN899382	JX091381	KF741964	KM023291
T. pittii	CBS 139.84 T = IMI 327871	JN899325	KJ865728	KJ885275	KM023297
T. pseudostromaticus	CBS 470.70 T = ATCC 18919 = FRR 2039	JN899371	HQ156950	KJ885277	KM023298
T. ptychoconidius	DAOM 241017 T = CV 2808 = DTO 180-E7	FJ160266	GU385733	JX140701	KM023278
T. purpureogenus	CBS 286.36 T = IMI 091926	JN899372	JX315639	KF741947	JX315709
T. purpureus	CBS 475.71 T = ATCC 24069 = ATCC 52513 = FRR 1731 = IMI 181546	JN899328	GU385739	KJ885292	JN121522
T. qii	CBS 139515 T = DTO 410-D1 = AS 3.15414	KP765384	KP765380	KP765382	MN969164
T. radicus	CBS 100489 T = FRR 4718	KF984878	KF984599	KF984773	KF985013
T. ramulosus	DAOM 241660 T = CV 2837 = DTO 184-B8.	EU795706	FJ753290	JX140711	KM023281
T. reverso-olivaceus	CBS 140672 T = CGMCC 3.18195 = DTO 317-C3	KU866646	KU866834	KU866730	KU866990
T. ricevillensis	NRRL 62296 T	KX657343	KX657056	KX657249	KX657582
T. sayulitensis	CBS 138204 T = DTO 245-H1	KJ775713	KJ775206	KJ775422	MN969146
T. solicola	CBS 133445 T = DAOM 241015	FJ160264	GU385731	KJ885279	KM023295
T. stipitatus	CBS 375.48 T = ATCC 10500 = NRRL 1006 = IMI 39805	JN899348	KM111288	KF741957	KM023280
T. subtropicalis	NRRL 58084 T	KX657337	KX657060	KX657250	KX657531
T. tabacinus	NRRL 66727 T = EMSL 2174	MG182613	MG182627	MG182606	MG182620
T. tenuis	CBS 141840 T	MN864275	MN863344	MN863321	LT559084
T. thailandensis	CBS 133147 T = KUFC 3399	JX898041	JX494294	KF741940	KM023307
T. tiftonensis	NRRL 62264 T	KX657353	KX657129	KX657163	KX657602
T. trachyspermus	CBS 373.48 T = ATCC 10497 = IMI 040043 = NRRL 1028	JN899354	KF114803	KJ885281	JF417432
T. ucrainicus	CBS 162.67 T = ATCC 22344 = FRR 3462 = NHL 6086	JN899394	KF114771	KJ885282	KM023289
T. udagawae	CBS 579.72 T = FRR 1727 = IMI 197482	JN899350	KF114796	KX961260	MN969148
T. varians	CBS 386.48 T = ATCC 10509 = IMI 040586 = NRRL 2096	JN899368	KJ865731	KJ885284	KM023274
T. veerkampii	CBS 500.78 T = DTO 258-I8 = IBT 14845 = IBT 32648	KF741984	KF741918	KF741961	KX961279

Nematode Preparation

H. zeae was cultured on maize (Zhengda 619), and cysts were collected at 40 days after inoculation Cysts were processed as described by Mo et al. (2021). Cysts were hand-picked with a dissecting needle under a stereomicroscope (SZX2, ILLT, Olympus). The isolated cysts were immersed in 0.5% NaOCl for 3 min surface disinfection and then gently washed fully with sterilized distilled water to remove the NaOCl, and then crushed with a ground-glass homogenizer to release the eggs from the cysts. Eggs were put in a 30 µm aperture hatching sieve and nested in petri dish (6 cm diameter) containing 3 ml distilled water (He et al. 2020). Fresh J2s were collected on the day of the experiment for subsequent experiments.

Preparation of strain GX1 spore suspension

The strain GX1 was cultured on potato dextrose agar (PDA) in Petri dishes for 7 days at 25°C. The conidia suspension was prepared by flooding the dishes with 3 ml sterilized distilled water, the agar surface was scraped with sterile glass rods, and the suspension was collected in a sterile 10 ml Centrifuge tube. The suspension was mixed in a vortex mixer (MIX-30S, MIULAB) for 3 minutes at 3000 × g to disperse the conidia. Conidia density was assessed with haemocytometer, and then adjusted to 1 × 10⁸ cfu/ml.

Evaluation of strain GX1 parasitic ability on Heterodera zeae

The parasitic ability of the strains to H. zeae cysts was determined using the method described by Liang et al. (2018). Thirty sterile cysts were placed on the edge of the colony on the plate culture of strain GX1. The cysts were picked out at 3 days after incubation at 25°C, and surface disinfection with 0.5% NaOCl for 3 min and gently rinse with sterile water three times, then put it on a PDA plate with 5 cysts each plate. Petri dishes were incubated at 25°C and all experiments were performed in triplicate. The cysts were observed and photographed daily using a microscope (Stereo Discovery, V20, Carl Zeiss) after transfer to PDA until day 5 to calculate parasitism. The percentages of parasitism were calculated using the following formula: Parasitism = number of cysts parasitized / total number of test cysts × 100%. The cysts were picked out and removed surface hyphae after 5 days. The half of the cysts were transferred to the hatching sieve to observe whether J2s hatched; the remaining cysts were gently punctured with a dissecting needle to observe the cyst wall, eggs and J2s from the cysts and photographed with a microscope (Axio Imager, Z2, Carl Zeiss).

The parasitic ability of the strain to H. zeae eggs/J2s was tested in a 96-well plate in the dark at 25°C, with approximately 100 eggs/J2s and 200 µL spore suspension each well. Five concentrations of spore suspension were tested at 1 × 10⁸ cfu/ml, 1 × 10⁷ cfu/ml, 1 × 10⁶ cfu/ml, 1 × 10⁵ cfu/ml, 1 × 10⁴ cfu/ml, and sterilized water as control, with 4 replicates for each treatment. All experiments were performed in triplicate. The parasitism was recorded using an inverted microscope (ECLIPSE, Ti-S, Nikon) at 0, 3, 6, 9, 12, 15 d after exposure. The percentages of parasitism were calculated using the following formula: Parasitism = number of eggs (J2s) parasitized /total number of test eggs (J2s) × 100%.

Preparation of strain GX1 fermentation filtrate

The strain GX1 was cultured in PDA medium at 25°C for 5–7 days. A disk of 9 mm in diameter from the edge of the colony was transferred to 250 ml triangular flask containing 100 ml of 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). After that, 10 triangle flask replicates were incubated each week for 3 weeks in a MQD-S2R shaker (Minquan Instrument Co., Ltd., Shanghai, China) at 160 rpm and 25°C (He et al. 2020). After 3-week, fermentation broth from total of 30 conical flasks was then filtered using 0.22 µm Millipore filters (Whatman, Clifton, NJ, USA) and 1-week fermentation filtrate (1-WF), 2-week fermentation filtrate (2-WF), and 3-week fermentation filtrate (3-WF) were prepared.

Effect of spore suspension and fermentation filtrate on Heterodera zeae eggs hatching

The effect of spore suspension on H. zeae eggs hatching was tested in 96-well plate in the dark at 25°C. After adding spore suspensions, the final volume was kept at 200 µL and approximately 100 eggs per well. The spore suspension was evaluated at five concentrations: 1 × 10⁸ cfu/ml, 1 × 10⁷ cfu/ml, 1 × 10⁶ cfu/ml, 1 × 10⁵ cfu/ml, 1 × 10⁴ cfu/ml, and sterilized water as a control. The effects of different concentrations of fermentation filtrate on H. zeae eggs hatching were examined. Freshly eggs of H. zeae were treated with 2.5% (i.e., fermentation filtrate volume: sterilized water volume = 1:39), 5%, 10%, 20%, and 50% 1-WF, 2-WF, and 3-WF; and 20% Czapek medium and sterilized water were used as controls. Approximately 100 eggs and 200µL of different concentrations of fermentation filtrate was added in each well of 96-well plate (He et al. 2020). Additionally, all treatments were done in quadruplicate, and experiments were performed in triplicate. The initial number of eggs was counted, and the hatched J2s were recorded using an inverted microscope (ECLIPSE, Ti-S, Nikon) at 0, 3, 6, 9, 12, 15 d after exposure in the dark at 33°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%.

Nematicidal activity of spore suspension and fermentation filtrate on Heterodera zeae J2s

Freshly J2s were obtained on the day of the experiment and dispensed in a 96-well plate with approximately 100 J2s per well. After adding spore suspensions or fermentation filtrate, a final volume of 200 µL was maintained in each well. Spore suspension concentration was 1 × 10⁸ cfu/ml, 1 × 10⁷ cfu/ml, 1 × 10⁶ cfu/ml, 1 × 10⁵ cfu/ml, 1 × 10⁴ cfu/ml and sterilized water as control. The fermentation filtrate concentration was 2.5%, 5%, 10%, 20%, and 50% 1-WF, 2-WF, and 3-WF, 20% Czapek medium and sterilized water as control. All treatments were performed in quadruplicate, and each experiment was repeated three times. The number of dead nematodes was counted using a microscope (ECLIPSE, Ti-S, Nikon) at 24, 48, 72 h after treatment with the solutions, and morphological photos were taken at 24 h. Nematodes were considered dead when straight and stiff and did not move after transfer to sterilized tap water for 12 h, even when agitated with a fine needle (Wu et al. 2021). The test was conducted at 25°C in the dark, and the J2s mortality rate was calculated using the formula: Mortality = (number of dead J2s/total number of J2s) × 100%.

Greenhouse Experiment

The biocontrol efficacy of strain GX1 against H. zeae was evaluated under greenhouse conditions using two experiments: (1) The spore suspension experiment: maize seeds (Zhengda 619) were pre-germinated for 2 days in wet paper towels at 25°C, and one germinating seed was sowed in each pot (10 × 10 × 10 cm) containing 300 g of autoclaved soil. 20 mL of 1 × 10⁸ cfu/mL spore suspension was applied to the soil around the seedlings after sowing, and control seedlings watered with an equal volume tap water. Seedlings were inoculated with H. zeae eggs (1000 eggs/pot) or cysts (30 cysts/pot) after 3 days. (2) The fermentation broth experiment: Three days after transplanting germinated maize seeds into pots, each pot was inoculated with 30 cysts. Subsequently, 30 ml of 50% and 100% 2-WF were used in this experiment and applied in pots, 20% of Czapek medium and tap water were utilized as controls. 30 ml of the corresponding solution was applied again on the 7th day.

A randomized design with 5 replicates for each treatment group was used for the pot experiment. All materials were maintained after inoculation at 28–30°C in a greenhouse with a 14 h light and 10 h dark photoperiod. After 40 days inoculation with nematodes, plant height, root length, fresh root weight and root dry weight of maize were measured. The H. zeae cysts in the soil were collected and counted using methods as previously described (Wu et al. 2014). Twenty cysts were randomly selected from the collected H. zeae cysts, and then crushed with a ground-glass homogenizer to release the eggs from the cysts, and the number of eggs was counted to obtain the average value of eggs each cyst. Total number of eggs = total number of cysts × average number of eggs per cyst. Cysts reduction rate = (cysts number in control − cysts number in treatment)/cysts number in control × 100%. These two experiments were repeated twice.

Statistical Analysis

Results of repeated experiments were combined. Statistical analyses were performed using the 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 at p < 0.05 using the Fisher’s protected least significant difference (LSD) test. Before statistical analysis, the mortality (%) data were transformed with log10 (x + 1). All figures for statistical analyses were made using Sigma Plot 10.0 (SPSS Inc., Chicago, IL, USA).

Isolation and identification of strain GX1

The total 21 strains of Talaromyces with parasitic ability on the cysts were isolated from H. zeae cysts. Among them, strain GX1 with higher efficiency parasitic ability on the cysts than others, which displayed unique phenotypic characters and grouped separately from known species in the multigene phylogenetic analyses. It was further studied and described as a new species named Talaromyces cystophila.

Taxonomy

Talaromyces cystophila Y.X. Mo, H.Y. Wu, sp. nov.

MycoBank MB 845592. Figure 1.

Etymology: Latin, cystophila refers to its functions, isolated from H. zeae cyst.

Diagnosis: This species produces smooth, ellipsoidal conidia; moderately fast growth on CYA, MEA, YES, and OA at 25°C, and grow normally on CYA at 30°C and 37°C. In: Talaromyces section Trachyspermi

Typus: China, Guangxi, H. zeae cyst, 10 Dec 2020, isolated by Y.X. Mo, Y.R. Long, H.Y. Wu. (holotype HMAS 352273, from dried culture of ex-type CGMCC40002 on PDA). Ex-type culture CGMCC40002.

ITS barcode: OM835900. (Alternative markers: BenA = ON164851; CaM = ON164853; RPB2 = ON164852).

Colony diam, 7 d (mm): CYA 21–26; CYA 30°C 18–21; CYA 37°C 18–21; MEA 32–33; MEA 30°C 18–22; OA 18–22; YES 19–24.

Colony characters: CYA, 25°C, 7d: Colonies moderately deep, centrally protuberant or plane; margins entire; texture velvety and short flocculent; sporulation sparse, yellow-green; mycelium pale yellow to yellow; soluble pigments absent; exudates absent; reverse yellow at the center, pale yellow at edge. CYA, 30°C and 37°C, 7d: Colonies moderately deep, slightly sulcate; margins entire; sporulation sparse. MEA, 25°C, 7d: Colonies thin, in some isolates raised at the center and in some isolates plane; slightly sulcate, irregular edge; mycelium yellow green, texture floccose; sporulation moderately dense, conidia en masse pale green; soluble pigments absent; exudates absent; reverse yellow-green. YES, 25°C, 7d: Colonies moderately deep, slightly sulcate; margins entire; mycelium light yellow at the center, white at edge; texture floccose; sporulation absent to sparse, conidia en masse pale green; soluble pigments absent; exudates light yellow droplets; reverse yellow, slightly sulcate. OA, 25°C, 7d: Colonies deep, in some isolates raised at the center and in some isolates plane; margins entire; mycelium white and pale yellow; texture velvety to floccose; sporulation dense, conidia en masse dark green; soluble pigments absent; exudates clear droplets; reverse pale yellow to yellow.

Micromorphology: Conidiophores biverticillate; stipes smooth, 150–200 × 2.5-4 µm; metulae 4–8, appressed, 8–13 × 2–3 µm; phialides1-4, acerose, 10–12 × 1.5–2.5 µm; conidia smooth, subglobose to ellipsoidal, 2.5-3 × 2.5-3 µm; Ascomata not observed.

Distinguishing characters: Talaromyces cystophila is a phylogenetic different species that belongs to the Trachyspermi group (Fig. 2). This species is distinguished by the production of smooth and ellipsoidal conidia. It does not develop red pigments or red exudates on CYA, MEA, YES and OA. It grew unrestrictedly on CYA, MEA, YES and OA, and can grow well at 30℃ and 37℃ on CYA.

Molecular biological identification of strain GX1

To determine the phylogenetic positions of strain GX1, 66 species of Talaromyces were included in ITS, BenA, CaM, RPB2 sequences in GenBank were downloaded, and the concatenated ITS-BenA-CaM-RPB2 phylogenetic analyses with sequences from ex-types, and T. cecidicola CBS 101419 T was used as an outgroup. The concatenated data set was 2429 bp in length (ITS 610 bp; BenA 392 bp; CaM 576 bp; RPB2 851 bp). The Kimura 2 parameter with gamma distribution (+ G) was the most suitable model for ML of the combined alignments. For BI, the General Time Reversible + G + I (GTR + I + G) was most suitable for the ITS and CaM data set, while Hasegawa-Kishino-Yano + I + G (HKY + I + G) for BenA and RPB2 were most suitable. The tree topology of ML and BI did not show significant differences. Therefore, BI trees were used to represent phylogenetic. Data with a posteriori probability (pp) and a bootstrap value (bs) were displayed on the tree (Fig. 2). The phylogenetic tree revealed that strain GX1 belonged to the Talaromyces section Trachyspermi and formed a monophyletic clade with 89% bootstrap support; however it was so unique that no close relatives were found in the section. Based on its phylogenetic and morphological peculiarity, the species was described as a new species named Talaromyces cystophila.

Parasitic ability of T. cystophila GX1 on the cysts, eggs and J2s of H. zeae

Talaromyces cystophila GX1 exhibited very strong ovicidal activity as well as cysts parasitism in H. zeae (Fig. 3). At 3 days after inoculation, the parasitism rate was 91.11%, whereas cyst in the control were not parasitized by the fungus (Fig. 3D). After one day of cysts transfer to PDA, hyphae were observed to grow from the cyst (Fig. 3A). After 3 days, the whole cyst was surrounded by dense hyphae (Fig. 3B). On the 4th to 5th day, the hyphae on the surface of the cyst formed a sporulating structure and produced large numbers of conidia (Fig. 3C). Eggs inside cysts parasitized by the strain have no J2s hatched, while J2s were hatched from the control cysts eggs on the 6th day. Microscopic observations revealed that hyphae of T. cystophila penetrated the cysts wall and colonized both sides of the wall (Fig. 3G-J). The inner superficial layer of the cyst wall falls off in fragments (Fig. 3K-L). The contents of J2s and eggs inside the cysts parasitized by T. cystophila appeared the dense vacuoles (Fig. 3E, I), and the probability of vacuoles was 65%-95%. However, the walls and eggs of control cysts appeared normal (Fig. 3M-O). Microscopic examination showed that the eggs and J2s were killed, but the eggs and J2s in spore suspension were not parasitized by T. cystophila GX1, although they are surrounded by spores. These results indicate that T. cystophila GX1 has an ovicidal effect on cysts and leads to structure destruction and tissue death.

Effects of T. cystophila GX1 spore suspension and fermentation filtrate on eggs hatching of H. zeae

Both spore suspension and fermentation filtrate of T. cystophila GX1 significantly inhibited the eggs hatching at different concentrations, and the inhibitory effect progressively increased with increasing concentrations of spore suspension and fermentation filtrate. In spore suspension treatment (Table 3), at 15 days after incubation, the cumulative hatching rates of the eggs in spore suspension of 1 × 10⁸ cfu/ml, 1 × 10⁷ cfu/ml, 1 × 10⁶ cfu/ml, 1 × 10⁵ cfu/ml, 1 × 10⁴ cfu/ml were 0.00%, 0.82%, 29.20%, 41.21% and 61.60%, respectively, significantly lower than controls (77.08%). In fermentation broth treatment (Fig. 4), at 15 days after incubation, the cumulative hatching rates in 2.5%, 5%, 10%, 20%, and 50% 1-WF were 27.51%, 19.88%, 12.74%, 1.07% and 4.14%, respectively, significantly lower than controls (Sterilized water 74.52%; 20% Czapek medium 73.30%). When comparing the different concentrations of fermentation filtrate at 1, 2, and 3 weeks, 20% 1-WF showed the strongest inhibition activity against the eggs hatching, and the hatching inhibition rate was 98.56%, which was significantly lower in comparison to controls (P < 0.05). Eggs in 20% 2-WF and 3-WF had the lowest cumulative hatching rates of 2.17% and 5.70%, respectively. The eggs in all treatments were decomposed to varying degrees using microscopic observation, and dense vacuoles formed in eggs. The eggs structure were damaged, and it could not develop normally and hatch (Fig. 5A, Fig. 6A), while the control eggs appeared normal to develop and hatch (Fig. 5B, Fig. 6B-C).

Table 3

Effects of different concentrations of *T. cystophila* spore suspension on the cumulative incubation of *H. zeae* eggs *in vitro* at 3, 6, 9, 12, and 15 d after inoculation
Concentrations (cfu/ml)	Time (d)
Concentrations (cfu/ml)	3	6	9	12	15
1 × 10⁸	0.00 ± 0.00 b	0.00 ± 0.00 d	0.00 ± 0.00 e	0.00 ± 0.00 e	0.00 ± 0.00 e
1 × 10⁷	0.00 ± 0.00 b	0.00 ± 0.00 d	0.00 ± 0.00 e	0.41 ± 0.41 e	0.82 ± 0.82 e
1 × 10⁶	2.52 ± 0.87 b	3.17 ± 0.74 d	17.70 ± 0.54 d	24.42 ± 1.33 d	29.20 ± 1.21 d
1 × 10⁵	18.74 ± 1.15 a	30.34 ± 1.15 c	36.90 ± 3.72 c	39.37 ± 2.99 c	41.21 ± 2.28 c
1 × 10⁴	17.80 ± 1.54 a	39.14 ± 0.93 b	48.77 ± 3.39 b	55.32 ± 2.53 b	61.60 ± 1.81 b
Control	19.75 ± 1.03 a	45.66 ± 1.89 a	58.57 ± 1.46 a	68.61 ± 3.02 a	77.08 ± 1.86 a
Values represent 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

Nematicidal activity of spore suspension and fermentation filtrates on Heterodera zeae J2s

Both spore suspension and fermentation filtrate of T. cystophila GX1 showed a significant lethal effect on H. zeae J2s, and the nematode mortality increased significantly with increasing concentrations of spore suspension and fermentation filtrate. There was a significant linear relationship between T. cystophila spore suspension and the J2s mortality (y_{24 h} = 0.646x – 3.733, r = 0.987, LC₅₀ = 5.95 × 10⁵ cfu/ml; y_{48 h} = 0.638x – 3.392, r = 0.984, LC₅₀ = 2.07 × 10⁵ cfu/ml; y_{72 h} = 0.607x – 2.849, r = 0.904, LC₅₀ = 4.97 × 10⁴ cfu/ml). The highest J2s mortality was observed in the spore suspension at a concentration of 1 × 10⁸ cfu/ml and reached 100% at 24 h (Table 4). The 2-WF demonstrated the best nematicidal action when compared to the 1-WF and 3-WF. When H. zeae J2s treated with 50% 2-WF, their mortality was 98.65% and 100% at 24 h and 48 h respectively (Fig. 6); After 72 h incubation, mortality of J2s in 2.5%, 5%, 10%, 50% 2-WF treatments reached 100%, which were all significantly higher than the controls (7.49%, P < 0.05). Specifically, microscopic observations revealed that when the freshly hatched J2s were added to 50% 2-WF, J2s immediately became inactive, bradykinetic and paralysis. At 12 h after treatment with spore suspension (1 × 10⁸ cfu/ml) or 50% 2-WF, the body of J2s gradually decomposed, generating vacuoles and constrictions, the J2s died with slightly bent and rigid bodies (Fig. 8A, Fig. 9A), while the J2s in the control group displayed normal activity (Fig. 8B, Fig. 9B-C).

Table 4

Effects of different concentrations of *T. cystophila* spore suspension on the activity of *H. zeae* J2s in vitro were measured at 24, 48, and 72 h after incubation
Time (h)	Concentrations (cfu/ml)	Mortality (%)	Linear regression equation (y = ax + b)	Related coefficient (r)	LC₅₀ (cfu/ml)
24	1 × 10⁸	100.00 ± 0.00 a	y = 0.646x – 3.733	0.987	5.95 × 10⁵
	1 × 10⁷	70.21 ± 0.66 b
	1 × 10⁶	51.70 ± 0.74 c
	1 × 10⁵	27.96 ± 0.97 d
	1 × 10⁴	18.12 ± 0.96 e
	Control	2.01 ± 0.71 f
48	1 × 10⁸	100.00 ± 0.00 a	y = 0.638x – 3.392	0.984	2.07 × 10⁵
	1 × 10⁷	83.95 ± 0.97 b
	1 × 10⁶	59.41 ± 0.76 c
	1 × 10⁵	39.96 ± 0.50 d
	1 × 10⁴	25.49 ± 1.01 e
	Control	4.89 ± 0.82 f
72	1 × 10⁸	100.00 ± 0.00 a	y = 0.607x -2.849	0.904	4.97 × 10⁴
	1 × 10⁷	97.05 ± 1.47 b
	1 × 10⁶	65.53 ± 0.99 c
	1 × 10⁵	52.29 ± 0.35 d
	1 × 10⁴	42.64 ± 0.53 e
	Control	6.05 ± 0.64 f
Values represent the mean ± standard error of six replicates per treatment using a combination of two different experiments. Mortality percent were transformed with arcsine before subjected to ANOVA and mean separation (Fisher’s LSD, p < 0.05). Means with the same letter were not significantly different (p < 0.05)

Greenhouse experiment

The effect of T. cystophila GX1 on H. zeae populations was evaluated using spore suspension and fermentation broth in greenhouse conditions. Results showed that both spore suspension and fermentation broth of T. cystophila GX1 significantly reduced the number of H. zeae densities. When the spore suspension (1 × 10⁸ cfu/ml) applied in pots inoculated eggs, the number of cysts and eggs decreased by 78.76% and 79.18%, respectively compared to control. Moreover, when the spore suspension (1 × 10⁸ cfu/ml) applied in pots inoculated cysts, the number of cysts and eggs decreased by 66.96% and 66.76% respectively compared to control (Table 5). In fermentation broth application experiment (Table 6), compared with the control, 50% fermentation broth decreased cysts and eggs by 56.17%, 53.16% respectively; and 100% fermentation broth decreased them by 77.47% and 76.06% respectively. Maize plant height, root length, fresh root weight, and dry root weight were not significantly different from the control (P < 0.05) after applied spore suspension and fermentation broth.

Table 5

Effect of *T. cystophila* GX1 spore suspension on nematode population and the growth of maize plants infected with *H. zeae*
Treatment	Plant height(cm)	Root length(cm)	Root fresh weight(g)	Root dry weight (g)	Number of cysts/ per pot	Number of eggs / per pot
spore suspension + eggs	26.17 ± 0.24 a	37.83 ± 0.33 a	5.36 ± 0.55 a	0.82 ± 0.08 a	90.33 ± 9.49 b	10556 ± 2420 b
tap water + eggs	26.43 ± 0.18 a	37.17 ± 2.73 a	5.59 ± 0.25 a	0.78 ± 0.04 a	425.33 ± 32.09 a	50698 ± 3746 a
spore suspension + cysts	26.67 ± 0.27 a	37.83 ± 3.44 a	4.77 ± 0.30 a	0.75 ± 0.61 a	88.00 ± 21.07 b	10742 ± 2418 b
tap water + cysts	27.00 ± 0.32 a	38.00 ± 2.89 a	5.78 ± 0.31 a	0.82 ± 0.79 a	266.33 ± 33.74 a	32320 ± 3816 a
Values represent means ± standard error of five replicates. Means with the same letter are not significantly different (p < 0.05) according to a Fisher’s protected LSD test

Table 6

Effect of *T. cystophila* GX1 fermentation broth on nematode population and the growth of maize plants infected with *H. zeae*
Treatment	Plant height(cm)	Root length(cm)	Root fresh weight(g)	Root dry weight (g)	Number of cysts/ per pot	Number of eggs / per pot
50% fermentation broth	23.67 ± 0.33 a	29.33 ± 0.67 a	7.99 ± 0.61 a	1.10 ± 0.12 a	67.00 ± 4.58 c	8633 ± 515 c
100% fermentation broth	23.33 ± 0.33 a	32.00 ± 2.31 a	6.15 ± 0.46 b	0.90 ± 0.21 a	130.33 ± 3.18 b	16893 ± 327 b
tap water	23.00 ± 0.58 a	29.33 ± 0.67 a	6.06 ± 0.21 b	0.83 ± 0.02 a	297.3 ± 8.11 a	36062 ± 734 a
20% Czapek medium	23.67 ± 0.33 a	28.00 ± 0.00 a	6.62 ± 0.13 b	1.03 ± 0.05 a	304.3 ± 3.76 a	37477 ± 715 a
Values represent means ± standard error of five replicates. Means with the same letter are not significantly different (p < 0.05) according to a Fisher’s protected LSD test

Plant-parasitic nematodes are one of the most important pathogens, and cause great economic losses by inflicting severe damage to crops and forests (Degenkolb and Vilcinskas 2016; Kiontke and Fitch 2013). Therefore, developing efficient and environment-friendly bio-agents is imperative to prevent or minimize plant nematode diseases. In China, there are few registered biological control products, especially biocontrol agents against plant nematodes. In this study, T. Cystophila GX1 isolates is capable of killing H. zeae eggs and J2s by penetrating the cyst wall, belonging to the section Trachispermi Yaguchi & Udagawa. The results suggest that this isolate has the potential to become an alternative to control for plant nematode.

The importance of the microbiome in facilitating nematode reduction has been observed in various crop systems (Adam et al. 2014; Elhady et al. 2018; Silva et al. 2021). Usually, multiple organisms and biocontrol mechanisms exist in the soil simultaneously to antagonize nematodes in the soil (Silva et al. 2018; Topalović and Heuer 2019). During the study period 2017–2020, H. zeae populations naturally declined. 60 strains abundant fungi isolated from H. zeae cysts and maize rhizosphere soils. Among of them, total 21 strains of Talaromyces are capable to parasitize the cysts. Talaromyces is an important genus in biotechnology and medical and food mycology. More than 177 species of this genus have been reported worldwide (Chen et al. 2021). About, 90% Talaromyces species reported distributed in warm temperate, subtropical, and tropical regions (Sun et al. 2020b). A monograph on Talaromyces applying a polyphasic species concept, including morphological, molecular and physiological characters, and accepted 88 species in seven sections: Bacillispori, Helici, Islandici, Purpurei, Subinflati, Talaromyces and Trachyspermi (Yilmaz et al. 2014), a new section Tenues was reported in 2020 (Sun et al. 2020a). The GX1 isolates had distinct phenotypic traits and categorize separately from other known species in the multigene phylogenetic analysis, and there are significant differences between this strain and other reported strains of the same genus, which produces smooth, ellipsoidal conidia without red pigments or red exudates on CYA, MEA, YES and OA. It grew unrestrictedly on CYA, MEA, YES and OA, and can grow well at 30℃ and 37℃ on CYA. It was described as Talaromyces cystophila based on morphological characteristics and genetic analysis.

Nematophagous microorganisms are natural enemies of nematodes and provide a promising approach for controlling nematode pests in nature. Some of these microorganisms create traps to capture and kill the nematodes from the outside. Others act as internal parasites, producing toxins and virulence factors that kill the nematodes from the inside (Li et al. 2015). Aspergillus niger can parasitize both cysts and eggs, and induced systemic resistance against plant-parasitic nematodes (Abd-Elgawad and Askary 2018; Jin et al. 2021). Similarly, Pochonia chlamydosporia usually parasitizes the eggs and adult females of plant-parasitic nematodes (Kerry and Crump 1977), disintegrate eggshell’s vitelline layer, as well as the partial dissolution of the chitin and lipid layers (Abd-Elgawad and Askary 2018). Our results suggested that T. cystophila had strong parasitic and lethal effects on H. zeae cysts with 91.11% parasitism rate. Interestingly, the inner superficial layer of the cyst wall fell off in fragments, the eggs inside cysts parasitized by T. cystophila cannot hatch normally, bodies of eggs and J2s appeared dense vacuoles. The eggs and J2s were destroyed and killed although T. cystophila cannot parasitize them. Similarly, Paecilomyces lilacinus produce proteases and chitinases to degrade nematode eggshell, displayed large vacuoles in the chitin layer, split vitelline layer and had lost its integrity (Khan et al. 2004). Trichoderma secretes extracellular hydrolytic enzymes such as trypsin-like protease PRA1 (Suarez et al. 2004), serine protease SprT (Chen et al. 2009), and chitinolytic enzymes chi18-5 and chi18-12 (Szabo et al. 2012). These extracellular hydrolytic enzymes enable the fungus to parasitize nematodes eggs and juveniles. In this study, we speculate that T. cystophila GX1 secretes some kind of enzymes or nematicidal substance, leading to eggs and J2s be destroyed, and ultimately developmental termination and died.

Talaromyces assiutensis is an efficient nematophagous species that exhibit predation and nematicidal activity against Meloidogyne javanica (Farhat et al. 2022; Hamza et al. 2017). Our results indicated that T. cystophila GX1 could predate cyst, and its fermentation filtrate and spore suspension inhibited eggs hatching and toxic to nematodes in vitro, and dense vacuolar in body of eggs and J2s. Similar phenomenon observed by He et al. (2020), where 1,5-Dimethyl Citrate hydrochloride ester from the fermentation filtrate of Aspergillus japonicus ZW-1 cause wrinkling on the surface of J2s, and dense vacuolar areas in the cytoplasm. Previous studies have shown that Talaromyces has unique and specific extrolites such as duclauxins, glauconic acids, mitorubrins, monascins, purpactins, rubratoxin, vermicellins etc. (Frisvad et al. 2013; Samson et al. 2011). So T. cystophila GX1 might produce the metabolites and enzymes destroy eggs and J2s, and severely affect their physical vitality. Therefore, further research is needed to separate and screen the nematicidal active substances produced by T. cystophila GX1 and the mechanism of killing nematodes, etc.

Talaromyces species have numerous benefits for the plants; they have been used as biocontrol agents for various pathogens (Tian et al. 2020, 2021). Our pot experiments also demonstrated that the spore suspension and fermentation broth of T. cystophila GX1 efficiently decreased H. zeae populations in the maize. Moreover, this fungal species have not negatively affect maize growth. This fungal species is still not evaluate against other plant parasitic nematodes, may be it exhibit even more strong nematicidal effect against them. The outcomes from this study revealed that GX1 is useful bio-control agent, could provide the fundamental foundation for the development of valuable and marketable biocontrol agent. Additionally, in the future this research will aid the researchers and farmers to use T. cystophila GX1 as an alternative to harmful chemical nematicides.

In conclusion, strain GX1 isolates from H. zeae cyst is a novel species described as Talaromyces cystophila. It had a strong parasitic ability on the H. zeae cysts, and its fermentation filtrate and spore suspension exhibited markedly inhibitory hatching on eggs and nematicidal activity on eggs and J2s. The pot experiments confirmed that both spore suspension and fermentation filtrates suppressed significantly the reproduction of H. zeae. T. cystophila is a soil inhabitant, which adapts to the soil environment condition. Therefore, in terms of innocuousness and sustainability, T. cystophila GX1 is a promising biocontrol strain for plant parasitic nematodes. Its parasitic and toxic effects on other plant-parasitic nematodes should be discussed in future. Especially, isolation and extraction of nematicidal substances and application in agricultural production.

Ethical Approval

This article does not contain any studies with human participants performed by any of the authors.

Competing interests

The authors declare that they have no conflict of interest.

Authors' contributions YXM and HYW conceived and designed the research; material preparation and data collection were performed by YXM and YZK; analyses were performed by YXM and XTC; the first draft of the manuscript was written by YXM and HYW; final drafting and critical revision of the manuscript were done by HYW, AS and LMJ. All authors contributed to the manuscript preparation (data analysis and/or writing).

Funding

This work was supported in part by the Natural Science Foundation of Guangxi Province (2020GXNSFDA297003), the National Natural Science Foundation of China (32160627) and Innovation Project of Guangxi Graduate Education (YCSW2022085).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Acknowledgements The authors thank Yan Rong Long for fungi isolation from cysts and soil.

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Novel ectophytic nematode antagonistic fungi and its potential application for nematode biocontrol

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Abstract

Figures

Introduction

Materials And Methods

Sample collection and strain isolation

Morphology of strain GX1

DNA extraction, PCR amplification and sequencing

Phylogenetic analysis

Nematode Preparation

Preparation of strain GX1 spore suspension

Preparation of strain GX1 fermentation filtrate

Greenhouse Experiment

Statistical Analysis

Results

Isolation and identification of strain GX1

Taxonomy

Molecular biological identification of strain GX1

Greenhouse experiment

Discussion

Declarations

References

Additional Declarations

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