zyxw Study of the nematocidal properties of the culture filtrate of the nematophagous fungus Paecilomyces Zilacinus Jean-Claude CAYROL, Caroline DJIANand Lucette PIJAROWSKI INRA, Station de Recherches de Nématologie et de Génétique Moléculaire des InverteZvés, 123, bd Francis-Meilland, B.P. 2078, 06606 Antibes Cedex, France. SUMMARY The culture filtrates of Paecilomyces lilacinus (nematophagous fungus) growing on different liquid media and under various conditions showeda very inconstant toxicity towards nematodes. The best nematocidal activity was obtained on malt medium with motionless aerated cultures. Light had no effect on toxin production. Although it was only generated at pH 5, the toxin had an activity independant of the pH. The activity ofthe toxic metabolite was very specific. When tested on seventeen species of nematodes it was only efficient against the Heteroderidae family (Meloidogyne, Heterodera). The mechanism involved in the toxin activity appeared as a neurotropic one because it had a reversible effect when the treatment period was less than 48 h. zyxwvut zyxwvutsrq Étude des propriétés nématicides du filtrat de culture du champignon nématophage Paecilomyces lilacinus Les filtrats de culture du champignon nématophage Paecilomyces lilacinus présentent une toxicité très variable vis-à-vis des nématodes selon les milieuxet les conditions de cultuiesutilisés. La plus grande activité nématicide a été obtenue sur milieu au malt en cultures stablesaérées sans influence de l’éclairement. Bien que n’étant produite qu’au dessous de pH 5, la substance toxique a une activité indépendante du pH. Cette substance toxique paraît tres spécifique. Testée envers dix-sept espkces de nématodes, elle ne se révèle réellement active qu’envers les représentants de la famille des Heteroderidae (Meloidogyne, Heterodera). Le mode d’action de cette substance semble de type neurotrope car sonesteffet réversible si la durée du traitement ne dépasse pas48 heures. T h e toxic effect of culture filtrates of different fungi on Meloidogyne larvae hasbeenstudied previously (Sakhuja, Singh & Sharma, 1978; Mani & Sethi, 1984; Dahiya & Singh, 1985). Recently, DechechiGomes Carneiro (1986) has demonstrated that a toxic substance killing the eggs of M. arenaria was present in the culture filtrate of the nematophagous fungus Paecilomyces lilacinus. The objective of the present work was to observe the activity of the culturefiltrate of P.’lilacinuson adults and larvae of different species of nematodes (phytophagous, mycophagous, saprophagous,entomophagous) and to study the conditions necessary to obtain the maximum nematocidal effect of the filtrate. Material and methods ORGANISM Nematodes Seventeen species of nematodes were tested forP. lilacinus activity. The origins of these species and thestages used for the biological tests are indicated in Table 1, col. 1 to 3. BIOLOGICAL TESTS After varied periods of incubation on different media, the culture filtrate was obtained by filtering it firstly through sterilized Whatman no. 2 filter paper which removes mycelium and secondly through 0.22 pm millipore filter avoiding spore contamination. The filtrate thus collected was tested pure orvariously diluted in sterile distilled water (1/2; 1/4; 1/8; 1/10; 1/20; 1/30). The tests were done in 24 well Nunclon plates; each well received 1 ml of the test filtrate (pure or diluted) to which about 50 nematodes were added in 20 pl of water. Each treatment was replicated four times. Toxicity was estimated according to the mean percentage of paralysed nematodes. Nematodes were considered paralysed if they did not move when probed with a fine needle. Two controls were used for comparison : zyxwvutsrqpo zyxwvutsrqp Fungus T h e fungal culture P. lilacinus used in the present investigation originated from the Amazonianarea of Brazil (Dechechi Gomes Carneiro, 1986). Revue Nénzatol. 12 (4) :331-336 (1989) 33 1 zyxwvutsrq zyxwvutsrq J. C. Cayrol, C. Djian & L. Pijarowski one in pure distilledwater, the other in the uninoculated sterilized culture medium always corrected to the same p H value as the tested culture filtrate. The reversible effect of the culture filtrate toxicity was also estimated according to the following method : the nematodes whose mobility was inhibited after various immersion periods in the filtrates were collected and dippedinfreshwater. The proportion of paralysed nematodes was estimated after 24 h in water. Table 1 Effect of the culture of Paecilomyces lilacinus on seventeen species of nematodes. Nematodes tested Biological group Species PHYTOPHAGOUS I Origins Aphelenchoides besseyi" A. fragariae" A. ritzemabosi" Meloidogyne arenaria"" M. incognita"" M. javanica"" Heterodera rostochiensis"" SPECIES In vitro SPECIES zyxwv zyx From roots Fungi cultures medium agar on SPECIES * ** *** Cultures on (Nigon, 1949) Cephalobus parvus" Panagrolaimzls mycophilus" mediumagar axei" Rhabditella Pristionchus lheritieri" ENTOMOPHAGOUS Neoplectana bibionis" N. calpocapsae * N. glaseri" 90 87 61 100 a 100 a 100 a 100 a cultures MYCOPHAGOUS avenae*Aphelenchus SPZCIES Aphelenchoides subtenzris" Ditylenchus myceliophagus" SAPROPHAGOUS zyx zy Mean percentages of paralysed lalvae afcer 20 h of dipping in pure culture filtrate""" on ' 18 b 21 b 2 zyxw oc Ic 82 50 Cultures Galleria caterpillars (Douenel, 20 1988) 30 b 25 b zyx Adults and larvae. Second-stage larvae. Ten day-old filtrate collected from montionless aerated cultures constantly exposed to light. Meansfollowed by the same letter in the column are not significantly different (P = 0.05) according to the Student's test. Number of replicates : 4. CULTURE MEDIA TESTED The growth of P. lilacinus isdependentfromthe nutritious aptitude of the culture medium (Villanueva & Davide, ,1984). As a general rule, the production of mycotoxins is alsogreatlyinfluenced by theculture medium (Smith & MOSS,1985). Consequently, the activity of the culture filtrate of P. lilacinus was tested after culturing the fungus on the following liquid media, al1 autoclaved at 1150 during 20 min before fungus inoculation : - media prepared with malt (15, 20, 25 and 30 g/1 of water). p H after steam sterilizationrespectively : 5.5; 5.47; 5.44; 5.40. - Czapek-Dox medium (Duncan, 1973). pH after steam sterilization : 6.4. - 332 - Czapek-Doxmodified medium(Naguib, 1960). p H after steam sterilization : 7.3. - Arconteil modified medium (Vey, Quiot & Vago, 1987). p H after steam sterilization : 6.5. - Mac Coy medium(Mac Coy, Hill & Cavanel, 1972). p H after steam sterilization : 6.9. The culture media prepared with malt were undefined media containing many unknown components present in malt. Nevertheless, these malt media have consistently shown improved toxin productionin a wide range of fungi (Smith & MOSS, 1985) and so were included in the tests. The four other media are synthetic. They differ only in theminera1 salts, the natureof the sugar (glucose or saccharose) andthe presence or absenceof iron salts. These fourmedia are commonly employed for culturing zy Revue Nématol. 12 (4) :331-336 (1989) zyxw Nematicidal properties of filtrate of Paecilomyces lilacinus zyxwvutsr zyx the toxin-producing fungi and it seems that their light differences between them are often sufficient to induce important variations in their toxic properties. Al1 the tested media were inoculated with P. lilacinus according to the following standard method : spores were used fromtwomonth-oldcultures of P. ZiZacinus in Petri dishes (100 mm diameter) on a standard agar medium (agar 15 g, malt 20 g/1 of water). Each Petri dish was filled with 20 ml of sterile distilled watergiving a spore suspension of about 3,2 x 107spores per ml. This spore suspension of about 3.2 x 107spores per ml. This spore 105 spores were inoculated per ml of liquid medium. The different liquid mediathus inoculated were kept in laboratory temperature conditions (22-270). non-aerated cultures (sterile air blown into the liquid medium by means of anaquariumpumpfor five minutes a day). EFFECTS OF THE PH Usingthemostperformantculturemediumand culture conditions, we have regularly measured the pH values of theculturefiltrateinordertoobservea possible correlation between p H and toxicity. Results INFLUENCE OF THE CULTURE MEDIUM CULTURE CONDITIONS TESTED The nematodes used to compare the toxicity between the eight different liquid media weresecond-stagejuveniles of M. arenaria which were the most sensitive to the toxicactivity of P. lilacinus. The results are givenfor 15 day-old filtrates collected from motionlessnonaerated cultures constantly exposed to light. Al1 the culturesof P. lilacinus were prepared in 500 ml erlenmeyer flasks filled with 300 ml of liquid medium (32 flasks for each tested medium). These 32 flasks were subdivided into four replicates of the following eight factorial treatments : 1) constant exposure to light or darkness, 2) motionless or shaked cultures, 3) aerated or Table 2 Influence ofthe culture medium on the activity of Paecilowyces lilacinus culture filtrates on Meloidogyne arenarialarvae (15 day-old filtrates collected from motionless non-aerated cultures constantly exposed to light)*. zyx zyxwvutsrqpon zyxwvuts Meut1 percentages of paralysed larvae afcer two hours of dipping Tested media Culture Malt Malt filtrate (15.20 gA) dilutions medium O 112 114 118 1110 * , (25.30 gA) Arconteil Czapek-Dox waodified medium 100 a 100 a OaX 100 a 100 a '97 a 100 a 1 ax 1 bx 37 b 52 b 2wc O cx Czapek-Dox Mac. Coy rnediunz 54 a 100 a 100 a laX 5 bx -----______---. 78 b 90 b 51 c 13 c medium modifed OaX O bx O dx O dx _____________------_-----------_-----OaX 1 bx 1dx O dx Means followed by the same letter in the columns and means underlined by the same line are not significantly different (P < 0.05) according to the Student's test. Number of replicates : 4. As confirmation of the general rule, thepresent results showed that thetoxic propertiesof P. lilacinusare greatly influenced by the liquid culture medium (Tab. 2). In Czapek-Dox modified medium the toxic metaboliteswere notproducedatanydilution. In Arconteil modified medium the toxicity was weak in pure culture filtrate (about 50 Yo of paralyzed larvae) and decreased very rapidly when the filtratewas diluted. In the two other synthetic media (Czapek-Dox and Mac Coy media) the toxicity was greater. The origin of a so markeddifference was possiblyrelated to the larger amounts of nitrogen and potassium in these two last media (respectively3 and 5 g of ammonium nitrate but only 0.7 g in the two firstmedia - 1 and 1.5 g of potassium but only 0.01 and 0.36 g respectively in Czapek-Dox and Arconteil modified media). zyxwvutsrq Revue Nénzatol. 12 (4) :331-336 (1989) 333 zyxwvutsrq zyxwvutsrq J. C. Cayrol, C. Djian & L. Pijarowski The highest toxicity was obtained in the malt composed media irrespectiveof the malt concentration.The main point concerning these malt media was to avoid filtering the liquid medium after autoclaving in order not to eliminate the brown flocculent always present at the bottomof the flasks. The existence of this flocculent was absolutely necessary for the toxin production by the fungus and we assume that essential nutrients required for toxin production were present in this precipitate. dipping) was greatly influencedby the four other culture conditions (motionless or shaken cultures; aerated or non-aerated). As ageneralrule, the toxic activity of P. ZiZacinus was better in motionless cultures than in shaked ones. It also appears that aerated cultures were more active than thenon-aerated ones. Finally, the best toxin production was obtained with motionless aerated cultures where the Meloidogyne larvae were al1 paralysed when dipped two hours in the greatest tested dilution (1/30)in a 15 day-old culture filtrate. zyxwvuts zyxwv zyxwvuts INFLUENCE OF THE CULTURE CONDITIONS T h e culture medium used for these tests was malt medium (15 g/l). The nematodes used were M . arenan'a larvae. Toxin production by the fungus was similar in light or dark conditions. In contrast, the results in Figure 1 show that the rate of toxicity (expressed after the maximum filtrate dilution in which al1 larvae were paralysed after two hours of Dilutions ofthefiltrate 1/30 INFLUENCE OF THE PH OF THE MEDIUM Fortheseexperiments,malt medium (15 g/l)in motionless aerated cultures exposed to light was used. The nematodestested were M . arenaria larvae. The results in Figure 2 show that the increase of the toxic properties of the culture filtrate (expressedafterthe greatest filtrate dilution in which al1 larvae were paralysed after two hours of dipping) were related to the decrease of the pH. In 15 day-old filtrate, the Meloido- Dilutions of thefiltl 3te 1/30 values 1/20 1/20 1/IO 1/10 1/8 I 18 1/4 / IO e 12 15 Age of the culture(days) zyxwvutsrqponmlkji Il2 O z zyxwvu Fig. 1. Influence of the culture conditionson the nematocidal activity of Paecilomyces lilacinus culture filtrates on Meloidogyne arenaria larvae. (The graph shows the filtrate dilutions where the larvae are totally paralysed accordmgthe to different culture conditions : O = aerated motionless cultures; O = non aerated motionless cultures; A = aeratedshaked cultures; A = non aerated shaked cultures). 334 114 I 2 8 4 I 6 8 1012 Age of the culture(days) 15 Fig. 2. Relationshipbetween pH and the toxicactivity of Paecilomyces lilacinus culture filtrates on Meloidogyne arenan'a larvae. (The graph shows the filtrate dilutions where Meloidogyne arenan'a juvenilesaretotallyparalysed) (A = pH values; O = filtrate dilutions paralysing the larvae). Revue Neinatol. 12 (4) :331-336 (1989) zyxw zyxw Nematicidal propertiesof filtrate of Paecilomyces lilacinus zyxwvut zyxwvutsrqp zyxwvut gyne larvae were al1 paralysed in the greatestfiltrate dilution(1/30)whenthe pH value was the lowest (pH 3.7). In order to observe if the activity of the culture filtrate was strictly limited to low pH, the pH of the culture filtrate was raised up to different p H values (5, 6, 7,8, 9)by adding somepl of 0.01 N sodium hydroxyde (without toxic effect on Meloidogyne larvae). A 10 day-old culture filtrate obtained in the same medium and in the sameconditions as previously described was used for this purpose. The different pH values ranging from 4.2 (natural filtrate) up to 9, were tested combined with various dilutions of the filtrate (O, 1/2, 114, 1/8) : no significative difference was observed between these various treatments. Two major informations are proceeding from these results : i) the increase of the toxin production was in inverse ratio to the decrease of the pH of the culture medium;it seems consequently thatthe toxin production by P. Macinus is moreimportantwhenthe fungus is in maximum growth period; ii) the so-produced toxin was able to act towards Meloidogyne larvae whatever was the pH of theculturefiltrate.Consequently we concludethatthe toxin production only occurs at low p H conditions but that the so-produced toxin acts in a wide range of pH values. 4 STUDY OF THE EFFECT OF THE CULTURE FILTRATE ON DIFFERENT SPECIES OF NEMATODES A 10 day-old culture filtrate produced in the same conditions as those described in the preceding experiment was used for this screening. The nematodes of the different species were immerged for 20 h in the culture filtrate. The results in Table 1, col. 4 show the mean percentages of paralysed nematodes for the different species. There were marqued differences in the toxicity of the culture filtrate according to the differentspecies of nematodes tested. The fourspecies belonging tothe family of Heteroderidae (Meloidogyne and Heterodera) were totally paralysed. Some others species were weakly affected (Aphelenchoides besseyi, A. fiagariae, A. ritzenzabosi and Rhabditella axei). There was no significant activity against the nine other species. zyxwvutsrqponmlkjih mean percentages IOOj tested were M. arenaria larvae. These larvae were dipped in pure culture filtrate during different periods (from 15 min to 72 h). The proportion of paralysed larvae present after 24 h in fresh water following different times in filtrate are presented in Figure 3. The toxic effect is clearly rapid since 97 O/o of larvae are already paralysed after only 30 minintheculturefiltrate. Secondly, the majority of larvae are able to recover the mobility in freshwater following dl treatment times up to 48 h.Only the 72 h treatment seems to kill the nematodes. m,.-.-.-.-.-.-. I Discussion 50- I The influence of the culture medium on toxin production wasvery important as thefiltratefromthe Czapek-Doxmodifiedmedium was inactive whereas maximum toxicity was observedwithmalt medium. Likewise, the toxic properties of the funguswere greatly influenced by theculture conditions. So the toxic metabolite was almost absent in shaked cultures very but activeinmotionlessaeratedcultures. Very different results have been observed with other fungi. For example, the entomogenous fungusMetarrhizium anisopliae gives the maximumtoxin production in shaked cultures Woberts, 1966). Conceming the age of the culture, the results show that the toxin production is more important when the fungus is in maximum growth period. That is also in contradiction with some others papers (Sakhuja, Singh & Sharma, 1978; Fargues & Robert, 1986) according to which mostof fungal pathogens produced toxins during their initial growth period. Hence it appears that the conditions necessary for mycotoxin production (medium, culture conditions, age) are very variable from one fungus to the other. Consequently when the ability of fungi to produce anytoxins are studied, it is absolutely necessary to testseveral parameters. Also to establishthe zyxwvut zyxwv I Q25 0 5 1 I 2 4 8 . I 24 4872 dippinq periods inculturefiItrate(hours) Fig. 3. Reversibility of the toxic effect of culture fiitrate of Paecilomyces lilacinus on Meloidogyne arenaria larvae.' (Ten day-oldfiltrate,collectedfrommotionlessaeratedcultures constantly exposed to light; = paralysed larvae in filtrate; O = still paralysed larvae in water.) zyxwvutsr STUDY OF THEREVERSIBILITY OF THETOXIC EFFECT A ten day-old culture filtratecollected from a motionless aerated culture on malt medium(15 g/l) exposed to light was usedfor this experiment. The nematodes Revue Nématol. 12 (4) :331-336 (1989) 335 zyxwvutsr zyxwvutsrq zyxwv J. C. Cayrol, C. Djian di L. Pijarowski identity of the toxic metabolite in the near future, it is absolutely necessary to define a well known synthetic medium which gives as good results as the malt medium. Indeed, the composition of the latteris too ill-defined to accurately consider the chemical analysis of the culture filtrate. The activity of the toxic metabolite of P. Zilucinus suggests a neurotropic mechanism. Firstly, the cuticles of stylet bearing nematodes are very impermeable; in the experiments, the activity of the toxin was tested by dipping the nematodes in the culture filtrate.Secondly, the paralysis of the susceptible species appeared very quickly (after 30 min of dipping) but the nematodes recovered their mobility when transfered to fresh water if the period of dipping did not exced 48 h. Al1 these results agree withthe hypothesis of a neurotropic action upon the nervous receptors of the nematode. However, these are preliminary findings, and, the exact mechanism of a such neurotropic action is yet to be established. These results are very different from those generally obtained when studying insect mycotoxinswhich act by injection of the culture filtrate in the insect hemocoele (Roberts, 1966) or sometimes by ingestion in mixture with the insect food (Robert& Fargues, 1986) but never by epidermic way. What is also interesting in these preliminary findings is the specificity of the toxic metabolite against a limited number of nematodes species. The reasons for such a specificity and the biochemical mechanisms of recognition involved on the nematode cuticle arealso of interest. This specificity appears as an additional problem in the nematodical mycotoxins research. It is clear that it is absolutely necessaryto test a lotof different nematodes species in order to reveal a possibletoxic activity. In the future, if mycotoxins areto be used as nematicides, their specificity is a real advantage. It enables only the Pest species tobe killed, withoutdestroying al1 the soi1 microfauna as current fumigants are doing. Thèse Parasitol., Univ. Sci. Techn. Languedoc, Montpellier, 119 p. DOUENEL, C.(1988).Nématodes-bactéries, une association efficace dans lalutte contre les insectes.Biofutur, 64 :39-42. DUNCAN, B. (1973).Nutritionand fat productioninsubmerged cultures of a strain of Penicillium lilacinum. Mycologia, 65 : 211-214. J. & Robert, P. H. (1986). Potentialités insecticides FARGUES, des destruxines mycotoxines produites par l’hyphomycète entomopathogène Metarrhizium anisopliae. I V Congr. Protect. Santé hum. et Cultures Milieu trop., Marseille, Juiz. 1986 :357-360. COY,C.W., HILL,A. J. & KANAVEL, R. F. (1972). A liquid medium for the large scale production of Hirsutella thompsonii in submerged culture. J. Invertebr. Pathol., 19 : 370. MAC zyxwvuts REFERENCES DAHIYA, J. 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K.; SINGH,1. & SHAW, S. K. (1978). Effect of SAKHUJA, some fungal filtrates on the hatchingof Meloidogyne incognita. Indian Phytopathol., 31 : 376-377. SMITH,J. E. & MOSS,M. O. (1985). Mycotoxins, Formation, Analysis. and Signifiance. Chichester, Wiley& Sons, 148 p. VEY, A., QUIOT,J. M. & VAGO, C.(1987). Mode d’action insecticided’unemycotoxine,laDestruxine E, sur les Diptères vecteurs et disséminateurs de germes, C. r. hebd. Séanc. Acad. Sci., Paris, 304, Sei. III :229-234. zyxwvutsrq zyx DECHECHI GOMESCARNEIRO, R. (1986). Étude des possibilités d’utilisation du champignon nénzatophage Paecilomyces lilacinus (Thom.)Samson, 1974, comme agent de lutte biologique contre Meloidogyne arenaria (Neal, 1889), Chitwood, 1949. VILLANUEVA, L. M. & DAVIDE, R. @. (1984). Influence of pH, temperature,lightandagarmedia on the growth and sporulation of a nematophagous fungusPaecilomyces lilacinus (Thom.) Samson. Philipp. Agric., 67 : 223-231. Accepté pour publication le 8 novembre 1988. 336 Revue Nématol. 12 (4) :331-336 (1989)