Host, temperature and media additive effects on the growth of Bursaphelenchus seani (') Robin M. GIBLIN andHarry K. KAYA Division of Nematology, University of California, Davis, Ca 95616, U.S.A.

SUMMARY Bursaphelenchus seani developed and reproduced on fourteen of eighteen fungi tested, includingtwo patho- genic fungi, Beauveria bassiana and Ascosphaera apis, and eight species of fungi isolated from adults and nests of its phoretic host, Anthophorabomboides stanfordiana. B. seani also developed and reproduced on alfalfa callus. Generation time (52 to 52)for B: seani on the fungus, Monilinia fructicola, was 16.0, 7.3, 4.2, 3.1 and 3.1 days at 15, 20, 25, 30 and 330, respectively. No development occurred at 9 and 360. At 250, B. seani dauer juveniles (JIII) were found after four weeks on cultures of M. fructicola. The mean number of B. seani produced per culture plate over time increased when potato dextrose agar (PDA) was supplemented with glycerol (115 mg/g hydrated PDA), oleic acid (10 mglg hydrated PDA) or lactic acid (6.7 mg/g hydrated PDA). JI1Is were found in supplemented cultures within two weeks and occurred in large numbers in glycerol-treated cultures after seven weeks.

RÉSUMÉ Efiets additifs de l'hôte, de la température et du milieu sur la croissance de Bursaphelenchus seani Bursaphelenchus seani s'est développé et reproduit sur quatorze champignons parmi les dix-huit testés, qui com- prenaient deux champignons pathogènes des insectes, Beauveria bassiana et Ascosphaera apis, et huit especes de champignons isolées à partir d'adultes et de nids de son hôte phorétique, Anthophora bomboides stanfordiana. Il s'est également reproduit sur des cultures de tissu de luzerne. La durée d'une génération (de52 à 52) pour B. seani, sur le champignon Monilinia fructicola a été de 16 ; 7,3 ; 4,2 ; 3,l et 3,l jours à 15, 25, 30 et 330 respectivement. Il n'y a eu aucun développement à 9 et 360. A 250, les a dauerlarven (5111)ont été trouvées après quatre semaines sur des cultures de M. fructicola. Le nombre moyen de B. seani produit par boîte de culture augmentait quand le milieu à la pomme de terre glucosé (PDA) étaitsupplémenté avec du glycerol (115 mg/gde PDA hydraté), de l'acide oléique (10 mg/g) ou de l'acide lactique (6,7 mg/g). Des JIII ont été trouvés dans les cultures supplémentées d8s deux semaines et se trouvaient en grand nombre dans les cultures traitées au glycerol après sept semaines.

The , Bursaphelenchusseani, is phoreti- Materials and methods callyassociated with the solitary soil-dwellingbee, Anthophorabornboides stanfordiana (Giblin & Kaya, HOSTRANGE STUDIES 1980 ; Giblin, Kaya & Brooks, 1981 ; Giblin & Kaya, 1983). Dauer juveniles (JIII) of B. seani are carried B. seani isolated from the reproductive tracts of in the adult bees' reproductive tract and are trans- adult A. bomboidesstanfordiana fromSonoma mittedto newhost cells duringoviposition. The County, California were subcultured on M. fructicola JIIIs moltto the propagative phase and feed on or Botrytiscinerea on potato dextrose agar (PDA). fungithat infest the bee cell. Fungalhost range Monoxenic stockcultures were established by studies were initiated to better understand thenema- inoculatingsurface sterilized onto one- tode-bee-fungirelationship andto select a fungus weelr-old M. fructicola culture. The nematodes were for mass rearing of B. seani. The ability of the nema- surface-sterilized in 1% wateragar containing the todeto grow on alfalfacallus andthe bacterium, fungicide, Aretan ' (methoxyethylmercury Escherichia Coli, was tested. The effects of tempera- chloride),and a bactericide,dihydrostreptomycin tureon the generation time of B. seani grown on sulfate (U. S. Biochemical Corp., Cleveland,Ohio), the fungus, Monilinia fructicola, were observed and for 48 h (Moody, Lownsbery & Ahmed, 1973). the effects of the addition of lactic acid, oleicacid A variety of fungi were isolated into pure culture or glycerol to the M. fructicola growth medium on and identified from the inner ce11 surfaces, provisions B. seani were examined. andbody surfaces of A. bornboidesstanfordiana

(l) Submitted for partial fulfillment of the Ph. D. for R. M. Giblin.

Revue Nématol. 7 (1) : 13-17 (1984) 13 R.M. Giblin & H.K. Kaya from Bodega HeadState Park, Sonoma County collebted andadded to individual culture Wellsof and Point Reyes National Seashore, Marin County, a multiwell tissue culture plate containing M. fruc- California (Tab. 1). In addition, certain saprophytic ticola mycelia.Cultures were incubated at each of fungiand plant and insect pathogenic fungi were seven temperatures (+ 10) : 9, 15, 20, 25, 30, 33 and includedin the host range studies. Petri dishes 360. Nematodes werecollected individuallyfrom (100 mm x 15 mm) with 15 ml of PDA were inocu- threerandomly selected culture Wells foreach lated with a small block of fungalmycelia (except temperature at6-24 h intervals until J2s of the next Meiarhiziumanisopliae and Beauveriabassiana generationwere first observed. Two trialswere whichwere inoculated bystreaking conidia). The done at 9,15, 20, 25, 30 and 33 andone trial for fungi were incubatedfor one weekbefore being 360. If development was not observed within three inoculated with B. seani. Nematodes were extracted weeks, thecultures were transferred to 250 and from stock cultures on a Baermann funnel, surface- incubated for oneweek and checked for development. sterilizedand counted. Surface-sterilization for the Generationtime is defined asthe time from 52 host range studies and medium additive experiments eclosion to 52 eclosion of the following generation. wasdone in two steps ; firstas described above (Moody,Lownsbery & Ahmed,1973) and sterilized MEDIUM ADDITIVES AND POPULATION GROWTH again as described byHara, Lindegren and Kaya OF B. seani (1981) for 3 h.Three hundred nematodes were pipetted ont0 an individual fungal culture,or uncon- Glycerol (115 mg/g hydratedPDA) or oleic acid taminated PDAor nutrient agar (NA) plate (control). (10 mg/g hydrated PDA) were added to PDA prior The number of nematodes was counted from three to autoclaving at. 15 p.s.i. at 1100 for 20 mn. Lactic randomlyselected Petri dish cultures at oneweek acid (6.7 mg/g hydrated PDA) was added aseptically intervals for four weeks. Nematodes were extracted toPDA after autoclaving. Petri dishes (100mm from individual plates for 3 h on a Baermann funnel x 15 mm)with ca 15 ml of supplementedPDA andthe total number of nematodes/plate was or plainPDA (control) were inocultatedwith M. estimatedby counting a 1 mlaliquot sample of 'a fructicola mycelia andincubated. After one week lrnown volume of the extract. thecultures were inoculated with 300 surface- The bacterium, E. Coli, wasassayed for its suit- sterilized B. seanilplate andincubated at 250. Two ability as a host for B. seani. E. Coli was streaked groups of threerandomly chosen plateswere ont0 NAplates and cultured for three days before harvest,edand counted, as previouslydescribed, inoculationwith 300 surface-sterilizednematodes. at 2, 4, 7 and12 weekintervals. Nematodes from Culturetime, extraction and counting procedures each of the different treatments or control cohorts wereas described above. Alfalfa callus was also for a particularweek were pooled after harvesting testedfor its suitability as a host for B. seani. and counted. Nematodeswere heat-killed and stained Alfalfaseeds, Medicagosatiua cv. Moapa,were in 45% acetic orcein for ca 24 h (Hirschmann, 1962). surface steriIized in concentrated H,SO, for 30 mn Arandom sample of 100stained nematodes were and rinsedwith sterile distilled water (Tamura examinedto quantify the appearance of JIIIs, as & Mamiya, 1975). Seeds were germinated on sterile determinedby the development of thegonad and filterpaper, soaked with sterilized water, in Petri other morphological features (Giblin & Kaya, 1983) dishes.Two germinated seedlings were aseptically intreated us. controlcultures. Mean numbers of transferredtoindividual Petri dishes (100 mm B. seani for this experiment were statistically com- x 15 mm)containing a growthmedium (Reidel, paredby a Kruskal-Wallistest (non-parametric, Foster & Mai,1973). Thecallus was incubated for single factor analysis of variance) and separation of seventeendays beforebeing inoculatedwith 100 meanswas done with the Wilcoxon and Wilcox surface-sterilizednematodes. Alfalfa cultures were test(non-parametric multiple range comparison harvestedand nematode populations counted 5, test). 7 and 10 weeks after inoculation as described above. Al1 tests were conducted at 25 -& 10. A host.is Results defined as anorganism capable of sust.aining the growth and reproduction of B. seani. HOSTRANGE STUDIES Eight species of fungi, isolated from the inner ce11 TEMPERATUREAND GENERATION TIME OF B. seani surface,provisions and bodie,s of A. bomboides B. seani eggs were obtainedfrom one-week-old stanfordiana servedas hosts for B. seani (Tab. 1). M. fructicola/B. seani cultures and washed in distilled However, the fungi thatsupported the highest water.Six to ten newly-hatched nematodes were nematode populations were not from bee cells. For

14 Revue Nérnatol. 7 (1) : 13-17 (1984) Host, temperature, media and growth of Bursaphelenchus seani

example, the plant pathogenic fungi, M. fructicola, EFFECTSOF TEMPERATURE ON GENERATION TIME B. cinerea and B. allii, andthe insect pathogenic OF B. seani fungi, B. bassiana and Ascosphaeraapis, sustained excellent growth and reproduction. B. seani did not Nematodes that were stored at 90 for three weelrs reproduce or reproduced poorly on the fungi, Cerato- did notdevelop, but went through one generation cystisallarztospora, M. anisopliae,Trichoderrna sp. withinone week after being transferred to 250. and Graphiumpenicilloides, andthe bacterium, E. Nematodesdid not survive incubation at 360 for Coli. Alfalfacallus was anadequate host, although three weeks. Thegeneration times of B. sean! Peakpopulations were not observeduntil seven cultured at 15,20,25,30 and330 were 384h (16 days), weeks after inoculation. The largest average nema- 175h (7.3 days), 101 h(4.2 days), 74 h (3.1 days) tode populations were observed on fungi two weeks and 74 h (3.1 days), respectively. afterinoculation, except sp.which was observed at three weeks. EFFECTOF ADDITIVES TO PDA ON POPULATION GROWTH OF B. seani Table 2 summarizes the effects of media additives Table 1 on the mean number ofB. seani produced per culture plateat 250. B. searzi grownon unsupplemented Mean populationnumbers of Bursaphelenchusseani media was not significantly different when compared on various fungi, Escherichia Coli to glycerol and oleic acidsupplemented cultures and alfalfa callus duringweek 2.After two weeks, supplemented cultures weresignificantly more productive than unsupplemented (control) cultures. Food source Highestpopulation S.D. mean JIIIs of B. seani werefound insupplemented cultures atfour weeks, butnot foundin control cultures until seven weeks after inoculation (Tab. 3). Therewere similar percentages of JIIIs perplate 1. Monilinia fructicola 167 400 9 525 incontrol and glycerol supplementedplates for 2. Beauueriabassiana 158 600 58 742 weeks 7and 12, although the number of JIIIs 3. Ascosphaeraapis 52 740 26 205 perplate in glycerol supplemented cultures was Botrgliscinerea 32 910 7 433 4. muchhigher than in controls or the other treated 5. alliiBotrytis 31 200 6 373 media. 6. Chaetomiurn sp. (1) 54029 10 266 During weeks 4 and 7, in glycerol supplemented 7. Fusnriunz sp. (l) 14 530 10 172 cultures,alarge number of 52nematodes were 8. Torula sp. (2) 13 120 6 044 9. Agraphiurn nouum 11 280 5 204 observed that were larger than normal J2s. At the 10. Penicillium sp. (?) 10 591 5 658 J2-JI11 intermolt these nematodes were significantly largerthan the normàl 52-53 intermolt (Giblin & 11. Oidiodendron sp. (l) 28310 6 586 Kaya, 1983). 12. Fusariurn solarzi (1) 8 320 3 288 13.Unknown Zygomycete (l) 7 212 909 14. Medicago satiua var. Discussion Moapa (Alfalfa callus)6 360 2 100 16. Fusariurnoxysporum (1) 4 040 1 667 B. seani feeds on awide variety of fungi for growth 16. Ceratocystisallanospora 565 420 andreproduction. Al1 fungiisolated from A. born- 17. Metarhiziumarzisopliae 390 173 boidesstanfordiarza adultsand cells as well asthe 18. Trichoderma sp. 220 125 insect pathogenic fungi, A. apis (pathogenic to the 19. Escherichia Coli 150 31 honeybee, Apismellifera) and B. bassiana (patho- (Racterium) genic to the sweat bee, pura and many 20. Graphiumpenicilloides 75 85 otherinsects) (Batra, Batra & Bohart, 1971)are 21. PDA 75 40 good hosts for B. seani. Some of the fungi isolated from the cells of A. bomboides starzfordiarza, such as F. solani and F. osysporum are suspected facultative pathogens of bees (Batra,Batra QG Bohart,1971). (l) Fungusisolated from bee ce11 of Anthophora Thus, B. searzi is similar toother bursaphelenchs bomboidesstanfordiana. which also have relatively wide fungal host ranges (z) Fungus isolated from adult A. bomboides stanfor- (Townsend,1964 ; McGawley, Jones & Birchfield, diana. 1980).

Revue Nimatol. 7 (1) : 13-17 (1984) 15 R.M. Giblin h H.K. Kaya

Table 2 Effects of supplements to PDA on population numbers of Bursaphelenchus seani grown on the fungus, Monilinia fructicola at 250

W eek Control (PDA)Control Week Glycerol (l) acid Oleic (2) acidLactic (3) (x f S.D.) (x& S.D.) (fT & S.D.) (s f S.D.)

2 175 991 f 99427aA (4) 226327 f 74052 aA 250 306 f 92 365 aA 119 889 f 36 281 aB 4 50076 f 12 718bA 290250 -j= 64814 bB 204 675 f 49 134 aB 120 150 & 20 299 aC 7 12 750 f 5 473 CA 108 615 f 59749 cB 51 900 f 40 078 bB 77 775 f 21 509 bB

12 1 227 f 748 dA 162 410 f 102 040 acB ------(6) 21 671 f 15 707 CC

(l) 115 mg/g hydrated PDA. (2) 10 mg/g hydrated PDA. (3) 6.7 mg/g hydrated PDA. (4) Mean of two replicates with three plates per replicate. Means followed by different lower case letters in a column and by different upper case letter in a row are significantly different (p -= 0.01) according to the Wilcoxon and Wilcox test. (5) Data not included due to contamination. = mean numher of B. seani per Petri dish culture (100 mm x 15 mm).

Table 3 Appearance of dauer juveniles (JIII) of Bursaplzelenchus seani on cultures of Monilinia fruciicola at 250

Week Control (PDA) Glycerol (1) Oleic Acid (2) AcidLactic (3) % (% % (XI % (%) % (Z)

2 O (0) O (0) "O (0) O (0) 4 O (0) 3 (8 708) . 1 (2000) 1 (1 200) 7 10 (1 270) (16292) 15 5 (2476) 2 (1 556) 12 29 (356) 850) 27 (43 (4) 3 (590)

(l) 115 mglg hydrated PDA. (2) 10 mg/g hydrated PDA. (3) 6.7 mg/g hydrated PDA. (4)No data.

B. seani has a fast generation time under laboratory for B. zylophilus (Tamura & Mamiya, 1975). In conditionsand when present inlarge numbers nature, B. seani is probably restricted to fungi as a ( > 4 000) causes visible damage to cultures of host food source. fungi.Similarly, B. xylophilus inhibitsmycelial The media-additive experiments demonstratedthat growthand sporulation of thefungus, Gliocladium glycerol, oleic acidand lactic acid are capable of sp. (McGawley,Jones & Birchfield, 1980). Perhaps alteringeither the reproductive rates and/or the nematode feeding damage ta insect pathogenic fungi survivability of B. seani inculture. Interestingly, in A. bomboidesstanfordiana cellsisselectively glycerol-supplemented agar produced greater nema- advantageous to bees associated with B. seani. tode yields per plate over time than unsupplemented Although B. seani is a mycophagousnematode, controls or the other additives tried. Also, glycerol- it can utilize plant tissue such as alfalfa callus under supplementedcultures produced larger numbers specialconditions. However, it grows slowly on of JIIIs of B. seani than unsupplemented controls alfalfa callus. Thesedata are similar to those obtained or the other t,reatments. Thus, M. fructicola grown

16 Nèmatol. Reuue 7 (1) :13-17 (1984) Host, temperature, media and growth of Bursaphelenchus sean on PDA supplemented with glycerol canbe useful associate of Anfhophorabolnboides stanfordiana for mass-culturing of B. seani and/or for the produc- Cockerell,1904 ( : Anthophoridae). tion of large numbers of JIIIs. Revue NCmatol., 6 : 39-50. Fungi infesting the cells of A. bornboides stanfor- GIBLIN,R.M., KAYA,H.K. & BROOKS,R.W. (1981). diana have been found in the provisions and on the Occurrence of Huntaphelenchoides sp. (Aphelenchoi- bee ce11 walls (Giblin,Kaya & Brooks, 1981). The didae) and Acrostichus sp. (Diplogasteridae) in the source of nutrition for thesefungi is notknown reproductive tracts of soil nesting bees (Hymenop- tera : Apoidea). Nematologica, 27 : 20-27. and probably varies depending upon the species of fungus. Females of some species in the genus Anllzo- HARA,A.H., LINDEGREN, J.E.62 KAYA, H.K. (1981). phora (i.e., A. abrupta and A. bomboides) are known Monoxenic massproduction of the entomogenous nematode. Neoaplectana carpocapsae Weiser, on dog to supplement the provisions of and line their cells food /agar medium. U.S. Dept. Agric., Sci. and Educ. with glycerides fromtheir Dufour’sgland (Norden Administr., W. Reg., Adv. agric. Technol., AAT-W- et al., 1980). Accordingly,components in the ce11 16, 8 p. lining and provisions may influence the physiology HIRSCHMANN,H. (1962). The life cycle of Ditylenchus andultimately the nutritive value of thefungus triformis (Nematoda : Tylenchida) with emphasison for nematode growth. Dauer juvenile (JIII)produc- post-embryonicdevelopment. Proc.helminth. Soc. tion in B. seani may be correlated with the presence Wash., 29 : 30-43. of bee-produced glycerides or fatty acids. MCGAWLEY,E.C., JONES,J.P. & BIRCH~~ELD,W. (1980).Reproduction of Bursaphelenchuslignicolus ACKNOWLEDGEMENTS onfungi isolatedfrom B. Zignicolus infectedpine J. : We thank Dr. A. R. Maggenti, University of Cali- trees. (Abstract). Nematol., 12 231. fornia, Davis for readingthe manuscript, thepersonnel MOODY, E. W., LOWNSBERY,B. F. & AI-IMED,J. M. of Point Reyes National Seashore and Bodega Head (1973). Culture of the root-lesion nematode Praly- State Park, California,for permission to collect and lenchus vulrzus on carrot disks. J. Nematol., 5 : 225- study A. bomboides stanfordiana and to Dr. T. Matsu- 226. motoand Mr. D. Supkoff, California Department of NORDEN,B., BATRA,S. W.T., FALES, H.M., HEFETZ,A. Food and Agrïculture for providing fungal cultures and & SHAW,G.J. (1980). Anthophora bees : Unusual for the identification of fungi isolatedfrom A. bom- glycerides from materna1 Dufour’s glands serve as boides stanfordiana. larval food and ceIl lining. Science, 207 : 1095-1097. REIDEL,R.M., FOSTER,J.G. & MAI, W.F.(1973). A REFERENCES simplified medium for monoxenic culture of Praty- BATRA, L.R., BATRA,S.W.T. 62 BOHART,G.E. (1971). lenchus penetrans and Ditylenchus dipsaci. J. Nema- The mycoflora of domesticated and wild bees (Apoi- fol., 5 : 71-72. dea). J. ECansas entomol. Soc., 44 : 13-44. TAMURA,H. 62 MAMIYA Y. (1975).Reproduction of GIBLIN,R.M. & KAYA,H.K. (1980). The association Bursaphelenchus lignicolus on alfalfa callus tissues. of a nematode, Huntaphelenchoides sp. (Aphelen- Nematologica, 21 : 449-454. choididae) with the solitary soil dwelling bee, Antho- TOWNSEND, J.L. (1964). Fungus hostsof Aphelenchus phorabomboides stanfordiana (Anthophoridae : avenae Bastian, 1865. and Bursaphelenchusfungi- Hymenoptera). (Abstract). J. Nematol., 12 : 221. uorus Franklin and Hooper, 1962 and their attrac- GIBLIN, R.M. & KAYA,H. K. (1983). Bursaphelenchus tiveness to these nematode species. Can. J. Micro- seani n. sp. (Nematoda : Aphelenchoidea), a phoretic biol., 10 : 727-737.

Accepté pour publication le 2 juillet 1982.

Revue A’èmatol. 7 (1) :23-27 (1984) 17