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Bursaphelenchus Seani (') Robin M 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 insect 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 nematode, 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 nematodes 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 ture on 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, oleic acid 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 fungal mycelia (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 and one 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 hydrated PDA) 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 and incubated at 250. Two ability as a host for B. seani. E. Coli was streaked groups of threerandomly chosen plateswere ont0 NA plates and cultured for three days before harvest,edand counted, as previouslydescribed, inoculationwith 300 surface-sterilizednematodes. at 2, 4, 7 and 12 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 particular week 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
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