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Nematicidal effect of freshwater fungal cultures against the
pine-wood nematode, Bursaphelenchus xylophilus
J.Y. Dong, Z.X. Zhao, L. Cai, S.Q. Liu, H.R. Zhang, M. Duan and K.Q.
Zhang*
Laboratory of Conservation and Utilization for Bio-Resources, Yunnan University, Kunming,Yunnan, PR China, 650091 Dong, J.Y., Zhao, Z.X., Cai, L., Liu, S.Q., Zhang, H.R., Duan, M. and Zhang, K.Q. (2004).
Nematicidal effect of freshwater fungal cultures against the pine-wood nematode,Bursaphelenchus xylophilus. Fungal Diversity 15: 125-135.
Twenty-two filtrates and 13 water-soluble extracts of broken fungal mycelia from 130freshwater fungal cultures were found to be pathogenic to the pine-wood nematode,Bursaphelenchus xylophilus following 48 hours exposure in vitro screening. The mobility ofover 90% of nematodes were inhibited by filtrates from Ophioceras commune (97.18%),Pseudohalonectria adversaria (96.49%), Pseudohalonectria lignicola (96.15%), Massarinathalassioidea (93.2%), Caryospora callicarpa (95.2%) and Annulatascus sp. (96.12%) and themycelia extracts from Helicomyces roseus (98.95%), Phomatospora berkeleyi (94.96%) andPseudohalonectria lignicola (95.59%). Aliphatic extracts of four freshwater fungal solid statefermentation products were found to immobilize over 50% of nematodes within a 12 hourexposure period at a concentration of 40 mg/mL. It was also observed that the aliphatic extractsof Pseudohalonectria adversaria, Xylaria sp. and Hyphomycete sp. were nematicidal, whereasMassarina bipolaris, Caryospora callicarpa and an unidentified strain were found to benarcotic in nature because nematodes revived when they were transferred to sterilized water.
When screening for nematicidal activities it is important to use approximate neutral and salineenvironments similar to the natural habitats of the test nematodes, as nematodes can be affectedby extreme pH and high osmotic pressure.
Key words: aliphatic extracts, broken mycelia extracts, filtrates, pathogenic
Introduction
Searching for new microbial strains as sources of biological nematicides is an important goal for those seeking to reduce the significant economicdamage caused by plant-parasitic nematodes. Fungi exhibit a range ofspecificities and modes of action in their antagonistic activities towardnematodes, offering an extensive pool of potential candidates to test (Siddiquiand Mahmood, 1996). Like other microbes, fungi can directly parasitize *Corresponding author: K.Q. Zhang; e-mail: [email protected] nematodes or secrete nematicidal metabolites or enzymes that affect nematodeviability. Toxic and inhibitory effects of several fungal filtrates have beenconfirmed (Desai et al., 1972; Alam et al., 1973; Sing et al., 1983; Khan andHussain, 1989; Khan and Kgan, 1992; Chattopadhyay and De, 1995; Pathakand Kumar, 1995; Sankaranarayanan et al., 1997). One interesting selectivenematicide reported recently is omphalotin, a cyclic dodecapeptide isolatedfrom Omphalotus olearius. Omphalotin is very potent against the plant parasiteMeloidogyne incognita (LD50: 0.75ug/mL). It is considerably more active than the commercially available nematicide ivermectin (Mayer et al., 1997; Stemeret al., 1997).
The objectives of this study were to determine the pathogenic effect of freshwater fungi isolated during our study on fungi on submerged wood instreams (Cai et al., 2002; 2003) on the pine wood nematode, Bursaphelenchusxylophilus.
Materials and methods
Fungi
One-hundred and thirty fungal strains (most belonging to the genera Annulatascus, Camposporium, Caryospora, Cyathus, Diaporthe, Dictyosporium, Dyrithiopsis, Eutypa, Eutypella, Leptosphaeria, Massarina,Nectria, Ophiobolus, Ophioceras, Phomatospora, Pseudohalonectria,Savoryella, Torula and Xylaria) were isolated from the submerged woodysubstrates collected in various freshwater habitats (e.g. Cai et al., 2002, 2003).
All strains were maintained on potato-dextrose agar slants.
For submerged cultivation, fungi were inoculated into 250 mL Erlenmeyer flasks each containing 70 mL medium (2% soybean power, 2%glucose, 0.2% peptone, 0.5% starch, 0.2% yeast cream, 0.4% NaCl, 0.05%K2HPO4, 0.05% MgSO4·7H2O, 0.2% CaCO3) and incubated for 10 days at 200 rpm at 26ºC on a rotary shaker. The culture filtrates and the water-solubleextracts of broken hyphae respectively served as stock solutions for screeningnematicidal activity. When the pH of the submerged cultures was over 8 orunder 5 the mobility of the nematodes were adversely affected. The influenceof pH on nematicidal properties was considered when adjusting the stocksolutions to neutral with NaOH and HCl solutions. Freshly prepared sterilizedmedia were clarified by centrifugation and designated as a control.
For solid-state cultivation, fungi were inoculated into 1000 mL Erlenmeyer’s flasks containing wheat medium. Wheat medium was preparedby soaking wheat seed in tap water for 3 days. The water was decanted, and ca.
200 g of wheat was packed loosely in individual Erlenmeyer’s flasks. A cotton plug was added and the flask was autoclaved twice at 15 psi for 40 minutes.
Mycelial plugs (1cm) were transferred to the flasks from the growing marginof the fungal colony on PDA plates and incubated at 27ºC till the funguscolonized the entire mass of wheat seed. The contents of flasks weretransferred to a shallow tray and lyophilised. The dried fungal hyphae wasground to powder using a mortar and pestle. The dried powder was soakedwith CHCl3 and MeOH solvents (1:1, v/v), maintained at room temperature for a week and filtered. The solvents were removed in a Vacuo at temperatures notabove 70ºC and the aliphatic extract of fungal mycelia was obtained and driedin a desiccator. A fixed amount of the well-dried extract was dissolved inDMSO, the concentration of which never exceeded 3% in the tested solutionand was diluted with water containing 0.3% (v/v) Tween-20 to prepare stocksolution of 40mg/mL. The same amount of DMSO dissolved in watercontaining 0.3% (v/v) Tween-20 was used as a control.
Nematodes
Botrytis cinerea was cultured on potato dextrose agar in Petri-dishes (diam. 90 mm) at 26ºC. Petri-dishes with fully grown fungus were inoculatedwith Bursaphelenchus xylophilus and left until fungal mycelia were completelyconsumed. The cultured nematodes (mixed stage) were separated from theculture medium by the Baerman funnel technique and enumerated on a gridunder a microscope (× 20). An aqueous suspension of the nematode (ca.15 000nematodes per mL) was prepared by appropriate dilution for use as a workingstock.
Bioassay of nematicidal activity
The nematotoxin bioassay was made in 6 cm Petri-dishes. Three hundred nematodes in 20 µL of aqueous suspension was transferred to Petri-dishescontaining 2 mL of fungal extracts and gently mixed. All dishes wereincubated at 25ºC. The numbers of live and inactive nematodes were countedunder a binocular microscope after different incubation times. Nematodes wereconsidered dead if they gave no response to physical stimuli such asmechanical stirring and pricking with the point of a needle. Toxicity wasestimated according to the mean percentage of dead nematodes. Each treatmentwas replicated four times and the data obtained analysed.
The revival rate of immobilized nematodes after treatment by fungal aliphatic extracts was also established. After 36 hours, the nematodes whosemobility was inhibited in the extracts were transferred to tubes and centrifugedat 10000 rpm. The supernatant fluid from each tube was removed with a pipette leaving about 20 µL. Sterilized water was added to make a total volumeof 1.5 mL. The tubes were again centrifuged at 10000 rpm. The supernatantfluid was removed and the nematode-containing solution remaining at thebottom was pipetted into a Petri-dish containing 1 mL sterilized water. Theproportion of revived nematodes was estimated after 12 hours in water.
Where there were no apparent effects on the nematodes during the exposure periods, the fungi were usually omitted from further studies.
Experiments were treated in triplicate for the fungi that appeared to immobilizeover 50% of nematodes.
Results and discussion
Toxic effect of submerged fungal cultures on Bursaphelenchus xylophilus
Over 50% of Bursaphelenchus xylophilus individuals were immobilized in 22 fungal filtrates (Table 1). After 48 hours, over 90% of nematodes wereimmobilized in filtrates of Ophioceras cummune (97.18%), Pseudohalonectriaadversaria (96.49%), Pseudohalonectria lignicola (96.15%), Massarinathalassioidea (93.2%), Caryospora callicarpa (95.2%) and Annulatascus sp.
(96.12%).
Over 50% of Bursaphelenchus xylophilus were immobilized in a solution of 13 broken fungal mycelia (Table 2). After 48 hours, a maximum death of98.95 percent of individuals were recorded using isolates of Helicomycesroseus, followed by Pseudohalonectria lignicola (95.59%) and Phomatosporaberkeleyi (94.96%).
The nematicidal effect of on nematodes can be group into five categories In the first category, the nematicidal effects occurred in both crude filtrates and water-soluble extracts of broken hyphae. When the pH of the testextracts were adjusted to pH 7.0, their was decreased in immobilization. Forexample, the crude filtrate of Ophioceras commune immobilized 52% ofnematodes within 48 hours of exposure, but the same filtrate at pH 7.0immobilized 0.63% of nematodes. It is thought that extremes in acidity oralkalinity result in the death of B. xylophilus because nematodes requireappropriate pH values for survival. Therefore, when extracts are tested fornematicidal activity, the pH value of the extracts are important.
Table 1. Effect of fungal filtrates on the pathogenicity of Bursaphelenchus xylophilus in vitro.
Name of fungi
Exposure time (fungal filtrates) in hours
In the second category, the fungi had no apparent effect on the vitality of B. xylophilus immersed in sterilized filtrates and hyphal extracts for periods upto 48 hours. However, when living nematodes were placed in neutral stocksadjusted with acid or alkali, many of them became inactive and appeared tohave been killed by substances in the filtrates. Immobilization of thenematodes may have been caused by high ion concentrations because B.
xylophilus may not survive in high ionic environments. For example, theneutral filtrate of Torula herbarum 123 immobilized up to 75% of nematodeswithin 48 hours, but the unadjusted filtrate immobilized only 23%. Thealkaline-treated hyphal extract of Ophioceras cummune 66 immobilized up to60% of nematodes, whereas the crude extract immobilized only 14% of them.
Therefore when the nematicidal activity of solutions is tested, osmotic pressureshould be considered and the activity should be evaluated in approximate ionicenvironments.
Table 2. Effect of fungal broken mycelia solution on the mortality of Bursaphelenchus
xylophilus in vitro.
Exposure time (fungal broken mycelia)
Name of fungi
Table 3. The five postulated circumstances on the nematicidal reasons of fungi.
Postulated reasons for
Number of fungi
immobilizing nematodes
filtrates
filtrates
belong to this type
alkaline environmentOver high osmotic pressure +: Over 50% of B. xylophilus were immobilized after 48 hours exposure time-: Less 50% of B. xylophilus were immobilized after 48 hours exposure time In the third category, the nematicidal effect only occurred in the filtrates and not in the broken fungal hyphal extracts. It was concluded that thenematicidal effect resulted from toxic substances secreted outside the fungalhyphae, i.e. exotoxins. Twenty-two fungal filtrates immobilized the nematodes,not only in crude filtrates but also in filtrates adjusted to pH 7.0. These fungiwere therefore subjected to further study. The most potent fungi were speciesof Caryospora, Diaporthe, Leptosphaeria, Massarina, Ophioceras andPseudohalonectria (Table 1).
Table 4. Effect of different fungal hyphae extracts on immobilization of B. xylophilus.
Exposure time (hours)
In the fourth category, the nematicidal effect only occurred in the broken hyphal extracts and not in filtrates. The nematicide is secreted within the fungihyphae (i.e. endotoxin). Natural and neutrally adjusted extracts of 13 fungalhyphae were pathogenic to the nematodes. They were species of Annulatascus,Caryospora, Cyathus, Ophioceras and Phomatospora. Among them, 8 strainsimmobilized up to 70% of nematodes within 48 hours exposure and 4 strainsup to 90% within 48 hours (Table 2).
In the fifth category, the nematicidal effect was most interesting because the fungal strains caused nematodes to die as a result of intracellular(endotoxins) and extracellular metabolites (exotoxins). Only three strainsproduced broken mycelia and culture filtrates that affected up to 50% of thenematodes within 48 hours of exposure. They were Helicomyces roseus,Pseudohalonectria lignicola and the unidentified fungus No.14 (Tables 1 and2).
Toxic effect of freshwater fungal solid-state fermentation products on B.
xylophilus
The effect of various extracts on the pine wood nematode is summarized in Table 4. The pathogenicity to nematodes did not always increase with lengthof exposure to the fungal extracts. After 12 hours the pathogenicity varied from5.3 to 97%. The maximum pathogenicity of 97% occurred in thePseudohalonectria adversaria isolate, followed by the Unidentified strain No.
1a (86.9%), Paraniesslia sp. (85.2%) and Massarina thalassioidea (65.6%).
No nematode mortality was observed even after 36 hours exposure in extractsof Camposporium quercilola, Diaporthe sp., Coelomycetes sp., Ophiobolus sp.,Paraniesslia sp. and Pseudohalonectria adversaria.
It was also observed that effects of culture extracts on nematodes mobility varied with length of exposure time. In the first example, as illustratedin the treatment of Camposporium quercilola, the nematodes were unaffectedand moved at each observation and activity levels did not diminish with time.
In second case the nematicidal activity of the culture extracts ofPseudohalonectria adversaria 129, Paraniesslia sp. 83, Caryosporacallicarpa, the unidentified strains No. 17 and No. 1a increased with exposuretime. In the third case, nematicidal activity decreased with time. For example,over 90% of nematodes were immobilized with initial exposure inPseudohalonectria adversaria 120 and 93 mycelial extracts, but at least 30%of those nematodes recovered after further exposure. In the fourth case, e.g.
extract of Massarina thalassioidea 50, the nematodes were initiallyimmobilized and became inactive after 24 hours of exposure, but recoveredduring the remainder of the exposure period. In the fifth cases, the number ofimmobilized nematodes was relatively stable from the start to the end ofexposure period.
The results of studies with Pseudohalonectria adversaria 120, 129 and 130 and Paraniesslia spp. 81, 83 and 84 were equivocal. The reasons for theseinconsistent results are unknown, however a possible explanation isintraspecific differences in the fungal strains. The various strains may producedifferent amounts and types of active metabolites.
Revival of immobilized nematodes
The number of revived nematodes was investigated for a further 12 hours in water in stock solutions (See Table 5). Results from strain No. 103,No. 17 and No. 5a showed almost all of the nematodes recovered their mobilityand became fully active after a further 12 hours in water, whereas extracts of strains No. 129, No. 128 and No. 1a appeared to kill the nematodes. Intreatments with strains No. 129, No. 128 and No. 1a, the nematodes did notrecover from exposure to extracts and less than 10% were active afterimmersion in water for 12 hours. Study of reversibility of the toxic effects offunal extracts indicate that Pseudohalonectria adversaria 129, Xylaria sp. 128,and Hyphomycetes sp. 1a were nematicidal, whereas Massarina bipolaris 5a,Caryospora callicarpa 103 and an unidentified strain No. D17 were possiblynarcotic in nature because nematodes revived when they were transferred tosterilized water.
Table 5. Results of the reversibility of the nematicidal toxin.
Strain No.
in aliphaticextracts)Pathogenicity (% 96.7 As compared to previous studies (e.g. Alam et al., 1973; Khan and Hussain, 1989; Chattopadhyay and De, 1995; Pathak and Kumar, 1995;Sankaranarayanan et al., 1997), this study has shown that nematicidal effectsof fungi extracts are unstable and the dead nematodes immersed in stocksamples can be revived with increasing exposure time. For example, the brokenmycelia solution of Ophiobolus sp. 56 immobilized the nematodes up to49.76% within 24 hours exposure period, but only 1.58% over a further 24hours exposure. The possible reason is that the toxicity of fungal metabolites istoo low to kill the nematodes, that is, the nematodes are immobilized by thesolutions, but recover with increasing time.
In conclusion, our results indicate that fungal extracts have potential to be developed as the nematicides, but the use of fungal extracts to control thepine-wood nematodes needs further study before being used as an alternative tochemical control. The main factors to be considered are fungal massproduction and phytotoxicity.
Why do freshwater fungi produce nematicides
In freshwater ecosystems, submerged woody substrata are main energy input (Wong et al., 1998). Wood is, however, a substrate greatly deficient innitrogen and therefore the nitrogen utilised by freshwater fungi may be obtained from other sources. Nematodes, are cosmopolitan organisms, adaptedto living in soils and water. They have been shown to be an integral part ofvarious ecosystems, serving as food for small invertebrates or fungi (Dropkin,1980). With their high nitrogen component, nematodes are thought to play animportant role in providing nitrogen to other organisms in freshwaterecosystems. Several nematophagous fungi have previously been reported fromwood submerged in freshwater, e.g. Dactylella ellipsospora Grove (Hyde andGoh, 1998) and Dactylella aquatica (Ingold) Ranzoni (Kane et al., 2002), andthese species are normally found from the dead bodies of the nematodes (K.D.
Hyde, pers. comm.). It would also make sense if other wood inhabiting fungioccurring on wood in freshwater were able to supplement their diets byobtaining nitrogen via digesting nematodes. The ability for these fungi toproduce nematicides that can kill nematodes, which they can subsequentlyconsume, would be advantageous. A pine-wood nematode, Bursaphelenchusxylophilus was therefore used in this study to establish whether freshwaterfungi can produce nematicides and the results are positive. Further studies arenecessary to establish whether freshwater fungi can kill and utilise nematodesin nature and whether any of these nematicides have biotechnological potential.
Acknowledgements
This study is supported by the National Natural Science Foundation of China (NSFC 30070006, 30230020, 3026002) and Yunnan Provincial Natural Science Foundation(1999C0001Z). K.D. Hyde is thanked for improving the English in this manuscript.
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(Received 18 November 2002; accepted 16 April 2003)

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