Haixiao Zhang · Zhiyan Wei · Jie Zhang ·Xuefeng Liu

Abstract Pine wilt disease, caused by Bursaphelenchus xylophilus and Bursaphelenchus mucnatus, is a serious quarantined disease. Arboreal nematode-trapping fungi of Pinus spp. are Effective predators on nematodes and have strong host adaptability. The development of these fungal resources may be an Effective way to control pine wood nematodes. We collected 515 samples of pine wilt disease from the areas of Ninghai City (Zhejiang province), Shuangbai County (Yunnan province), and Daxing’anling (Heilongjiang province),China. Through isolation, culture and identif ication, 6 species of nematode-trapping fungi ( Arthrobotrys cladodes r,A. oligospora, A. musiformis, A. dendroides, Dactylellina ellipsospora, Monacrosporium thaumasium) were identif ied for predation against B. xylophilus, and 9 species ( Arthrobotrys dactyloides, A. cladodes r A. oligospora A. dendroides,Dactylellina ellipsospora, Dactylella asthenopaga, D. leptospora, Arthrobotrys superba, Monacrosporium drechseri)were identif ied for predation against B. mucnatus. This study provides information in the classif ication of arboreal predator nematodes and provides an important basis for future biological control of pine wood nematodes.

Keywords Bursaphelenchus xylophilus · Bursaphechus mucnatus · Morphological classif ication · Nematodetrapping fungi

Introduction

Pine wilt disease is caused by the pine wood nematode,Bursaphelenchus xylophilus(Steiner & Buhrer) Nickle,resulting in rapid death of many species ofPinus(Cheng 1988). Since its discovery in 1905 and subsequent reporting in 1934, it has become widely distributed in Japan, South Korea, China, in North America and countries in Europe(Kiyohara and Tokushige 1971). The disease in China and Japan is the most serious (Zhu 1995) and in China, it has been listed as a severe disease for quarantine. Regardless of the growth rate of the species, or age and size, infected trees will die relatively quickly, leading to serious impact on forest economy and ecology, damage to natural landscapes (Zhang et al. 2013; Gao et al. 2015). Studies have shown that the pine wood nematode disease appeared in western parts of the country and rapidly spread to the north, currently the westernmost Sichuan Liangshan city, the northernmost part of Liaoning province, and in a number of counties (districts) of China. This disease has now spread across 14 provinces and cities, invading many national scenic area and key ecological areas, and has broken through the limit of annual average temperatures above 10 °C as suggested by traditional theory.The target of the pine wood nematode has expanded from the pastPinus massonianaLamb. andPinus thunbergiiParl.to otherPinusspecies and threatens the health of China’s nearly 60 million hectares of pine resources (Jiang 2019).

The pathogenic conditions of the pine wood nematode disease are more complex than those of many common plant diseases. Vector insects are required to carry the nematodes to a suitable pathogenic environment. In Asia, biological control of the disease is mostly focused on controlling the transmission of insect vectors, but nematode-trapping fungi also play an important role in controlling plant and animal nematodes and edible fungus nematodes (Manueli 1999). At present, more than 200 species of nematode-trapping fungi have been reported (Nordbring Hertz et al. 2011; Vidal-Diez de Ulzurrun and Hsueh 2018). Although these fungi have some Effect on controlling pine wood nematode disease,most come from soils and animals. It is diffi cult for them to adapt to the environment of a tree, which limits their application in controlling pine wood nematodes. Chemical controls are Effective but pollute the environment and destroy ecological balance. Therefore, it is important to develop an Effective biological control using arboreal nematode-trapping fungi. This study classif ies these fungi and f ills in the def iciency of the classif ication of arboreal predator nematodes. The objective is to provide a basis for the development of Effective biological reagents for the control of pine wood nematodes and pine wood-like nematodes.

Materials and methods

Sample collection and treatment

Bursaphelenchus xylophilussamples of pine wilt diseased tree roots, and residues were collected in pine wilt disease areas of Ninghai City, Zhejiang province;Bursaphelenchus mucnatussamples collected in Shuangbai County of Chuxiong City, Yunnan province and in the Daxing’anling area of Heilongjiang province. Three to four diseased trees and one healthy tree were randomly selected. Discs 3 cm thick were removed, beginning at ground level, and then every 1 m. A small disc was removed from the side branches at the junction with the trunk, and then a small disc every other year. Samples were numbered. For soil collection, sampling points were randomly located 1 m from the diseased tree,litter was removed and 10 gm of soil collected, numbered and stored refrigerated.

A total of 515 samples were collected, of which 382 samples (235 samples were from the trunk and 147 samples were from the branches) were collected from Zhejiang province,and the other 133 samples (80 samples from the trunk and 33 samples from the branches) from Heilongjiang and Yunnan.Samples were divided into two parts, bark and xylem; the bark was divided into epidermis, mesodermal, and endothelium and the wood divided into sapwood and heartwood. If blue stains were present, they were marked. Branch samples were divided into two the epidermis and the xylem.

Isolation and culture of nematode-trapping fungi

Saprophytic nematodes were inoculated into an oatmeal medium and cultured at 25 °C. After propagation, the nematodes are f lushed out with sterile water using the Baermann funnel method (Baermann 1917; Gray 1984) and diluted into a nematode suspension with a concentration of 500 pieces/mL.

The xylem of each sample was cut into small 8-10 pieces and the bark was cut; both were sprinkled on the CMA medium. The root samples were also sprinkled onto two rows of the medium. This was repeated three times and then the nematode suspension was added at 5000 nematodes per dish. The number and the date of separation were recorded.After reaching equilibrium, the culture plates were incubated at room temperature in the dark. After four weeks, the predator species were identif ied by microscope and counted;individual conidiospore of the arboreal predator nematode sinensis were selected for preservation.

CMA medium was prepared with 20 g of corn f lour,1000 mL of distilled water, and 15 g of agar, and Oatmeal medium was prepared with 10 g of oatmeal, 5 mL of distilled water.

Panagrellus redivivuswas derived from the pathology laboratory of forest protection disciplines of Northeast Forestry University.

Identif ication of fungi

Under sterile conditions, 1-2 cm × 0.5 cm agar blocks were removed from a pure culture plate of predatory nematode fungi and 1 mL of nematode suspension added to the culture dish. A nematode suspension of 0.5 mL was added to the surface of the culture medium to induce the production of predatory organs. After 48 h, the fungi were described morphologically and photographed. The isolation rate of each bacteria is equal to the number of dishes that the bacteria appear/the total number of dishes that are separated × 100%

Results

Fungi for predation against Bursaphelenchus xylophilus

Thirteen samples of pine wood nematode Fusarium fungus wilt were collected from Haining City, Zhejiang province;235 samples of trunk and 147 samples of branches were also collected. Two genera and six species of nematode-trapping fungi were identif ied:Arthrobotrys cladodesr,A. oligospora,A. musiformis, A. dendroides,Dactylellina ellipsospora,Monacrosporium thaumasium.

Arthrobotrys cladodes Drechsler (Fig. 1)

Arthrobotrys cladodesDrechsler, Mycologia. 29:463-467,1937 (Drechsler 1937)

Trichothecium cladodes(Drechsler) Soprunov F F., Predacious hyphomycetes and their application in the control of pathogenic nematodes. Academy of Sciences of the Turkmen SSR, Ashkhabad: 1-365. 1958 (Soprunov 1958).

On CMA medium, colonies of this fungus are white, the hyphae colorless and separated; the conidiophores are erect,unbranched or with 1-2 branches, length: 170-300 μm, 4-6 separated; tumorous protuberances at the top of conidia are usually enlarged, coralliform, 5-30 conidia, head-shaped,ovate to oblong, double-celled, axial cells and paraxial cells of the same length, 12.5-21.3 (average 17.5) × 4.5-7.5 (average 2.5) μm, the predator is a three-dimensional strain net.The strain is preserved in the Forest Pathology Laboratory of Northeast Forestry University, specimen number C31233.

Specimen collector: Wei Zhiyan.

Specimen collection place: diseased tree of black pine wilt in Ninghai City, Zhejiang province.

The original record (1937):conidiophores are branched,length: 54-200 μm, 10-20 conidia on a tumor node, elliptical to long oval, size 11.7-17.5 × 5.8-8.2 μm. Specimen C31233 is slightly longer and slightly narrower than the original record, but other characteristics are consistent, so it is recognized as this species.

Distribution in China:Yunnan, Guizhou and Heilongjiang.

Arthrobotrys oligospora Fres (Fig. 2)

Arthrobotrys oligosporaFres. Heft. 1(1): pl.1-9, 1850 (Fresenius 1850)

On CMA medium, the colonies of this fungus are white and grow vigorously, hyphae are colorless, separated and branched, the conidiophores erect or knee-shaped without branches, length 237.5-437.5 μm, tip width 3-5 μm, base width 5-8 μm, conidiophore 2-7 separate, small stalk tumorlike protrusion; conidial double cell, pear-shaped to obovate,separated at the constriction; size 17.5-23.8 (3.9) × 8.8-11.3(7.9) μm, the length of the distal axis is about twice the length of the paraxial cell; the predator is a three-dimensional strain net. The strain is preserved in the Forest Pathology Laboratory of Northeast Forestry University, specimen number D4122.Specimen collector: Wei Zhiyan.

Fig. 1 Arthrbotrys cladodes. 3, 6: Conidiophores, 1-2, 4-5, 7-11:Conidia, 12: Adhesive nets. Bars = 10 μm

Fig. 2 Arthrobotrys oligospora. 1: Conidiophores, 2-4: Conidia, 5:Adhesive nets. Bars = 10 μm

Specimen collection place: diseased tree of black pine wilt in Ninghai City, Zhejiang province.

The original record (1937):Colonies white to pale pink,conidiophores erect, stem length 180-470 μm, conidia size 17-31 (23) × 8.7-15.6 (13) μm, spores are slightly smaller than the original record.

Distribution in China:widely existing species.

Arthrobotrys musiformis Drechsler (Fig. 3)

Arthrobotrys musiformisDrechsler, Mycologia. 29: 481,1937 (Drechsler 1937)

Fig. 3 Arthrbotrys musiformis. 1-2, 7-11: Conidia, 3-5: Conidiophore, 6: Adhesive net. Bars = 10 μm

On the CMA medium, the hyphae are colorless and separated; conidiophores are colorless, erect, separated,unbranched; height is 200-500 μm, the base width 5-9 μm,and the tip width 2-4.5 μm; the conidia are colorless,elliptical, one separated, slightly curved, wide at the top and narrow at the bottom, 20-28.8 (average 23.5) × 5-8.8 (6.8)μm, the predator is a three-dimensional strain net. The strain is preserved in the Forest Pathology Laboratory of Northeast Forestry University, specimen number B3223.

Specimen collector: Wei Zhiyan.

Specimen collection place: diseased tree of black pine wilt in Ninghai City, Zhejiang province.

The original record:The conidiophores are 140-640 μm,the base 5-7.5 μm wide, the distal end 2.5-5 μm, and the conidia are 20-47.5 (30.9) × 7-12.5 (10.3) μm. Compared to the original record, the spores are slightly smaller.

Distribution in China:Anhui, Beijing, Guizhou,Guangxi, Hubei, Shandong, Sichuan, Yunnan, Tibet,Heilongjiang.

Arthrobotrys dendroides Kuthubutheen & webster(Fig. 4)

Arthrobotrys dendroidesKuthubutheen & Webster, Trans Brit. Mycol. Soc. 84: 563, 1985 (Kuthubutheen et al. 1985)

The colonies were white on CMA medium, the aerial hyphae sparse and colorless, branched and separated. The conidiophores are erect, solitary or aggregated into bundles; the spore bundles are 0.5 to 1.2 mm wide and about 1 mm long. The base of a single conidial stalk is 2 to 3 μm wide and 2 to 2.5 μm wide at the end. The upper part is tortuous, the middle part straight, and there are occasional branches, re-breeding; there are inconspicuous small odontoids, 1-7 divisions. Spores are born on small odontoid processes. Conidia are separated, located in the middle, short cylindrical, obtuse at the ends, gradually abaxially pointed,slightly curved, 12-15 (5.2) × 4.5-5.5 (2.8) μm, average 14.6 × 4.0 μm. A three-dimensional network was formed on CMA with nematode medium. The strain is preserved in the Forest Pathology Laboratory of Northeast Forestry University, specimen number E3134.

Specimen collector: Wei Zhiyan.

Specimen collection place: diseased tree of black pine wilt in Ninghai City, Zhejiang province.

Distribution in China:Yunnan province.

Worldwide distribution:China, the United States,Malaysia.

Da ctylellina ellipsospora (Preuss) M. Scholler, Hagedorn&A. Rubner, Sydowia 51(1): 110 (1999) (Fig. 5)

Monacrosporium ellipsosporum(Preuss) Cooke & Dickinson, Trans. Brit. Mycol. Soc. 48: 623, 1965 (Cooke and Dickinson 1965).

Menispora ellipsosporaPreuss, Sturm Deutschl. Fl. Abt.3: H.8: T.47, 1851.

Fig. 4 Arthrobotrys dendroides 1: Synnemata, 2-3: Conidiophores,4: Adhesive nets, 5: Conidia; Bars = 10 μm

Dactylella ellipsospora(Preuss) Grove, J. Bot. Lond. 24:200, 1884 (Grove 1884).

Dactylellina ellipsospora(Preuss) M. Scholler, Hagedorn& A. Rubner Sydowia 51(1): 110(1999).

On the CMA medium, the colonies are white to pink;the mycelium is sparsely transparent, separated and branched; the conidiophores are colorless and erect, height 187.5-325 μm, base width 5 μm, tapered to the top, top width 1.5-2.5 μm, with 2-6 septa; conidia solitary at the top of conidiophores, colorless, spindle-shaped, tapered at both ends; occasionally, there are distally rounded spores,3-4 divisions, with 4 divisions as the main, middle cells extra large, the shape of the waist drum, spore size 37.5-65(45.9) × 7.5-18 (15.6) μm; prey on nematodes with sticky balls. The strain is preserved in the Forest Pathology Laboratory of Northeast Forestry University, specimen number B521022.

Specimen collector: Wei Zhiyan.

Fig. 5 Dactylellina ellipsospora. 1-2: Conidiophores, 3-10: Conidia,11: Adhesive knobs. Bars = 10 μm

Specimen collection place: diseased tree of black pine wilt in Ninghai City, Zhejiang province.

Distribution in China:Hebei, Zhejiang, Anhui, Hubei,Yunnan, Hainan, Guangxi, Taiwan and Tibet.

Worldwide distribution:wide.

Monacrosporium thaumasium (Drechsler) de Hoog &van Oorschot (Fig. 6)

Monacrosporium thaumasium(Drechsler) de Hoog & van Oorschot, Stud Mycol. 26: 120, 1985 (Drechsler de Hoog and van Oorschot 1985).

Dactylaria thaumasiaDrechsler, Mycologia. 29: 522,1937 (Drechsler 1937).

Golovinia thaumasia(Drechsler) Mekht., Mikol. Fitopatol. 1: 276, 1967 (Mekhtieva 1967).

Candelabrella thaumasia(Drechsler) Rifai, Reinwardtia.7: 369, 1968 (Rifai 1968).

Fig. 6 Monacrosporium thaumasium 1: Conidiophores, 2-4:Conidia, 5: Adhesive. net Bars = 10 μm

Arthrobotrys thaumasia(Drechsler) S. Schenck, W.B.Kendr & Pramer, Can J Bot. 55: 984 (Schenck et al. 1977).

Mycelium is colorless, separated, branched; conidiophores are colorless, 4-8 separated, erect solitary, 120-400 μm long,base width 5-7.5 μm, tapered upward, top width 2.0-2.5 μm.It has a conidia at its tip and occasionally branches; conidia are colorless spheres to inverted pear shape, base is truncated,1-2 divisions, size 25-37.5 (30) × 15 to 22.5 (18) μm, there are large central cells; the worms are used to prey on nematodes.The strain is preserved in the Forest Pathology Laboratory of Northeast Forestry University, specimen number B01211.

Specimen collector: Wei Zhiyan.

Specimen collection place: diseased tree of black pine wilt in Ninghai City, Zhejiang province.

Distribution in China:Yunnan.

Worldwide distribution:China, the United Kingdom.

Fungi for predation against Bursaphelenchus mucronatus

In the Daxing’anling area of Heilongjiang province,A.cladodes,A. dendroides, andA. dactyloidesDrechsler were separated fromPinus sylvestrisvar.mongolicalarch. Among them,A. cladodesandA. dendroidesare the newly recorded species in Heilongjiang province. No predator nematode was isolated from QinglinP. sylvestrisvar.mongolica.

In Shuangbai County, Yunnan province, eight species of nematode-trapping fungi were separated from the diseased tree:A. dendroides,A. oligospora,A. cladodes,A. superba,Dactylellina ellipsospora,M. drechsleri,Dactylella asthenopaga,D. leptospora. Among them,Dactylella asthenopagais a newly recorded species in China.

Arthrobotrys dactyloides Drechsler (Fig. 7)

Arthrobotrys dactyloidesDrechsler, Mycologia. 29:486-487, 1937 (Drechsler 1937).

Dactylaria dactyloidea(Drechsler) Soprurunov, Predaceous hyphomycetes and their application in the control of pathogenic nematodes. Academy of Sciences of the Turkmen SSR, Ashkhabad: 1-365. 1958 (Soprurunov 1958).

The hyphae are colorless, branched, separated, and often fused. The large conidial spores are erect, unbranched or with few branches, 400-650 μm long, and gradually taper to the top. The base is 5-6.2 μm wide and the end is 1.5-2 μm wide. The conidia grow on the dentate stalk. There are two types of large conidia. Type A conidia are in the shape of thick sticks, sometimes curved, with a partition in the middle, and a very small number of 2 separated, conidia size 25-32.5 (29.1) × 4.5-7.5 (6.3) μm, average 42.1 × 7.5 μm.Type B conidia are ovate to pear-shaped, with a size of 10 to 16.3 (14.1) × 3.8 to 5 (4.8) μm, one separated. A shrink ring is formed on the CMA. The conidia can also produce a predator shrink ring on CMA medium without the addition of nematodes. Type B conidia are usually produced in large amounts after the induction of nematodes. The strain is preserved in the Forest Pathology Laboratory of Northeast Forestry University, specimen number YJ1232.

Specimen collector: Liu Xuefeng.

Specimen collection place: Daxing’anling, Heilongjiang province.

Distribution in China:Hebei, Beijing, Yunnan and Guizhou.

Worldwide distribution:Widely existing species.

Dactylella asthenopaga Drechsler

Dactylella asthenopagaDrechsler, Mycologia. 29: 498,1937 (Drechsler 1937).

Dactylariopsis asthenopaga(Drechsler) Mekh., Mikol Fitopat. 1:279, 1967 (Mekhtieva 1967).

Fig. 7 Arthrbotrys dactyloides 1-4: Conidiophore, 5-8: Conidia, 9:Constricting rings and trapping nematode. Bars = 10 μm

The hyphae are colorless, branched, and separated.Conidiophores are relatively high, 100-500 μm, usually 125-175 μm, with a conidium at the top and 1-2 short branches at the top; conidia are colorless, inverted conical or stick-shaped, base narrow and f lat, the tip obtuse,20-40 × 7.5-10 μm, the average is 31.8 × 8.6 μm, and there are 1-3 partitions; the sticky ball is caught on the CMA with nematode medium to catch the nematode. Research specimen number: X0222.

Specimen collector: Liu Xuefeng.

Specimen collection place: Shuangbai County, Chuxiong City, Yunnan province.

Dactylella leptospora Drechler (Fig. 8)

Dactylella leptosporaDrechler, Mycologia. 29: 507, 1937(Drechsler 1937).

Monacrosporium leptosporum(Drechsler) Rubner, Stud.Mycol 39:79, 1996 (Rubner 1996).

Dactylaria dasguptaeShome & Shome, Mycopath.Mycol. Appl 30:216, 1966 (Shome and Shome 1966).

Fig. 8 Dactylella leptospora. 1: Conidiophores, 2-4: Conidia, 5:Non-constricting ring, 6: Adhesive knobs. Bars = 10 μm

Kafi addinia fusariisporaMekht., Mikol. Fitopatol 12:8,1978 (Mekhtieva 1978).

Dactylella fusariispora-(Mekht.) Zhang Liu & Cao,Mycosystema 7:112, 1994 (Zhang et al. 1994).

Its hyphae are colorless, branched and separated,2.5-7.5 μm. Conidiophores erect, few branches, colorless, divided into 1-3 septa, length 20.0-50.0 μm,base width 1.5-2.5 μm, end width 1.0-2.5 μm; conidia colorless, long spindle shape to column shape, size 27.6-52.5 × 2.5-5.25 μm, average 39.1 × 4.7 μm, there are 3-8 partitions. When the conidia matures, one or two sticky spheres are formed at the end of the spore. The size of the sticky sphere is 7.5-8.0 × 7.5 μm, the length of the shank is 12.5-20.0 μm, the base of the stalk is 2.5-5.0 μm wide,and the width of the end is 2.0-2.5, divided into 1-2 compartments; forming non-shrinking rings and sticky balls to catch nematodes on CMA with nematode medium. Research specimen number: E4221.

Specimen collector: Liu Xuefeng.

Specimen collection place: Shuangbai County, Chuxiong City, Yunnan province.

Distribution in China:Beijing, Yunnan, Tibet, Guizhou and Hubei provinces.

Worldwide distribution:China, the United States, India,Russia, Uzbekistan.

Arthrobotrys superba Corda (Fig. 9)

Arthrobotrys superbaCorda Pracht-Flova Europaischer Schimmelbildungen 43 p. 1839 (Corda 1839).

Didymozoophaga superba(Corda) Soprunov & Galiulina, Mikorobiologia. 20:493, 1951 (Soprunov 1951).

Arthrobotrys drechsleriSoprunov, Predacious hyphomycetes and their application in the control of pathogenic nematodes. Academy of Sciences of the Turkmen SSR,Ashkhabad : 1-365. 1958 (Soprunov 1958)

Didymozoophaga kirghizicaSoprunov, Mikrobiologiya 20: 496, 1951 (Soprunov 1951).

Fig. 9 Arthrobotrys superba. 1-3: Conidiophores, 4-5: Conidia, 6:Germinating conidium, 7: Adhesive nets. Bars = 10 μm

Arthrobotrys kirghizica(Soprunov) ex Soprunov, Predaceous hyphomycetes and their application in the control of pathogenic nematodes. Academy of Sciences of the Turkmen SSR, Ashkhabad: 1-365. 1958 (Soprunov 1958).

The hyphae are colorless, branched, and separated.Conidiophores erect, unbranched, with multiple tumor nodes, 4-6 septa, 177.5-275.0 μm long, base and end of spore stalks are nearly thick, base width 4.0-5.0 μm, end width 2.0-5.0; conidia are born on the small stalk of the tumor, and 20-39 spores can be produced on one tumor.Conidia have a septate, located in the middle, no signif icant constriction at the septate, near ellipse, distal obtuse circle, size 12.5-17.5 × 5-7.5 μm, average 14.6-17.0 μm.A three-dimensional network is formed on the CMA with nematode medium to catch nematodes. Research specimen number: B2111.

Specimen collector: Liu Xuefeng.

Specimen collection place: Shuangbai County, Chuxiong City, Yunnan province.

Distribution in China:Anhui, Yunnan, Guizhou,Hainan, Tibet, Hubei and other provinces.

Worldwide distribution:wide.

Monacrosporium drechseri (Tarjan) Cooke &Dickinson (Fig. 10)

Monacrosporium drechseri(Tarjan) Cooke & Dickinson,Trans. Brit. Mycol. Soc 48: 623, 1965 (Cooke and Dickinson 1965).

Dactylella drechsleriTarjan, Mycopathlogia 14: 143,1961 (Tarjan 1961).

Golovinia drechsleri(Tarjan) Mekht., Mikol. Fitopatol 1:276, 1967 (Mekhtieva 1967).

The hyphae are colorless, separated, 2.5-5 μm wide; the conidiophores are colorless, erect, 2-5 septa, 112-137 μm high, 1-2.5 μm at the upper end, 2.5-3 μm at the lower end and a single conidium at the top, or forming 1-2 branches at the top, and a single conidium is formed on each. Conidia colorless, spindle-shaped, rounded at the top, 20.0-40.0 (31.1) × 7.5-17.5 (12.2) μm, 2 to 4 divisions, mainly 3 divisions; A sticky ball with handles to predate nematodes. The size is 5.0-10.0 × 5-7.5 (8.3) μm,the shank 7.5-15.0 μm, and the width is 2.0-2.5 μm; connected to the hyphae with an undivided handle. Specimen number: A0132.

Specimen collector: Liu Xuefeng.

Specimen collection place: Shuangbai County, Chuxiong City, Yunnan province.

Distribution in China:Yunnan, Guizhou and other provinces.

Worldwide distribution:China, the United States.

Fig. 10 Monacrosporium drechsleri. 1: Conidiophores, 2-4:Conidia, 5: Adhesive. net Bars = 10 μm

Key to Chinese species of nematode-trapping fungi

1. Conidiophore with denticulate loci (Arthrobotrys)-2.

1. Denticulate loci absent 0.6

2. Nematodes trapped by simple or complex adhesive network 0.3

2. Nematodes trapped by stalked constricting ringsArthrobotrys dactyloides.

3. Conidiophore bunchedArthrobotrys dendroides.

3. Conidiophore unbunched 0.4

4. Conidia obovate, repeated multiple fertilityArthrobotrys oligospora.

4. Conidia do not repeated multiple fertility 5.

5. Conidiospore curved, conidia ellipsoidal, 1 septate,born singly on sterigma like branches at conidiophore,22.5-33.0 × 6.1-8.5 μmArthrobotrys musiformis.

5. Conidiospore do not curved, conidia ellipsoidal,19-25 × 64-88 μm.Arthrobotrys cladodes.

5. Conidiospore do not curved, conidia ellipsoidal, 1 septate, located in the middle, the partition does not shrink signif icantly, 12.5-17.5 × 5-7.5 μmArthrobotrys superba.

6. Conidia spindle shaped with a considerably enlarged central cell. (Monacrosporium)-7.

7. Nematodes trapped by simple or complex adhesive network, conidia 1-3 septate, conidia born in a terminal group at apex of conidiophoreMonacrosporium thaumasium.

7. Nematodes trapped by stalked constricting rings,conidia 4 septate. 40-46 × 10-13 μmMonacrosporium ellipsosporum(Dactylellina ellipsospora).

8. Nematodes trapped by stalked constricting rings,conidia 3 septate. 20.0-40.0 × 7.5-17.5 μmMonacrosporium drechseri.

6. Conidia fusiform without enlarged central cell.(Dactylella)-9.

9. Nematodes trapped by adhesive knob, conidia 3-8 septate,Dactylella leptospora.

9. Nematodes trapped by adhesive knob, conidia 1-3 septateDactylella asthenopaga.

Discussion

Nematode-trapping fungi were isolated only from diseased trees, indicating that these species have a strong dependence on nematodes. Healthy pines are not suitable for nematode growth or the fungi have an inhibitory Effect, which limits their role in predation and control of nematodes. Some studies have pointed out that nematode trapping fungi are carried by vector insects to the trunk. However, in the separation of the Tianniu tunnel (Lun et al. 2019), only the spores are isolated and the separation rate is relatively low, so this pathway may not be the main one. Compared with predatory nematodes in soil under the same environment, pine wood nematode wilt disease has fewer arboreal nematode fungi species and low isolation rate. They are highly correlated in terms of species, so predation on trees is not excluded.Nematophagous may be derived from the soil. Predatory nematodes isolated from soil also have a strong predation Effect on pine wood nematodes.

At present, it has been reported that almost all of the fungi that prey on nematodes are from soils (Back et al. 2002).There are few species isolated from pine wood nematode infected trees. They are recorded as:A. oligospora,A. ellipsospora(Fukushige 1991). Judging from the progress of current research, the species of arboreal fungi that can prey on nematodes is considerable, and there is much potential.However, an analysis of existing data on the disease of pine wood nematodes reveals that there is a problem with the method of separation. Because arboreal predator nematodes are vulnerable in the fungal community, and are inhibited by other bacteria when nutrients are relatively good and begin to develop when soil nutrients are poor. Therefore,improved culture methods increase the chance of isolation from nematodes. The wilt disease of pine wood nematode is often accompanied by blue-straining, but nematode-trapping fungi are also isolated from the blue-negative sample. However, the diff erence is not as signif icant as compared with the non-blue meta-material. The correlation between the bluetransformed and predatory nematodes is not strong.

How to utilize these fungal resources to reduce damage or control of pine wilt blight is the focus of future research.These problems need to be further studied in the case of nematode-feeding processing, the release technology in the forest, and how to colonize and expand after entering the tree and the continuous control of the pine wood nematode.

Conclusions

This research showed that tree trunks may be rich in fungi of the nematodes. From 515 samples of disease trees, 11 species of three genera of arboreal predator nematodes were separated and identif ied. Six species of nematode-trapping fungi were identif ied for predation againstB. xylophilus,nine species of nematode-trapping fungi were identif ied for predation againstB. mucronatus.Among them,A. cladodes,A. oligospora, A. dendroides, andDactylellina ellipsosporaare the common species for predation against the two nematodes.

AcknowledgementThe author is grateful to colleagues who assisted in preparing this paper.

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