416 R ichard K. Be11jami11 ct al.

most autho ritative source, and few species have been lul ar ycastlike fo rms (Fig. 18.10 [ ')'n-tbiotaphrina and described since that work was completed. Couch undescribed taxa]) tl1 at inhabit speciali zed host ceU recorded the largest number of species (36) fr·o m the and coelo mic cavities, and the basidiomycetes and United tares, altho ug h these fungi probably arc pre­ ascomycetes associated with siricid wood wasps, ants do minantly tropical. Coll ectors in tropical regio ns likely and termite (Table 18.3). wi ll discover a large number of new taxa, a point Couch (1938:50) recogni zed when he stated that " no discus­ sion of geographic distribution will be of much va lu e." BARK BEETLES AND FUNGI Otl1er useful reference to Septobasidialcs arc those of Couch (1935) and Azema (1975 ). Fungi arc often associated with beetles that inhabit bark and wood of li ving or recently dead trees. Tho e TECHNIQUES FOR SAMPLING FUNGI fun gi fa ll into two categories. One category encompasse

INVOLVED IN GARDENING SYMBIOSES TABLE 18.3 Fungi Involved in Gardening Symbioses with Artlu-opods Mutualistic associations between insects and the fu ngi on whj ch tl1 ey feed o r fro m which they acquire enzymes for Ascomycota digesti o n often are referred to as gardening symbio es accharomycctcs (M. M . Martin 1987). Not all of the fungi in these asso­ Saccharomvcctalcs (Ascoidcn, Dipodnscus, Picbin, Cn11dida ) Pyrcno mycctcs ciations arc o bligate members of such symbioses; ratl1er Hypocrcalcs (undescribed ycastlikc forms associated with there is a continuum of associatio ns ranging fr·om those planthoppcrs) in which tl1e fung us is o nl y dispersed by the insect to Xvlarialcs those tl1at are true gardening associatio ns. Some of the X yla ri accac ( Xylnrin) Microascalcs associations are of intcrc t because they may provide Ccratocystiaccac ( Ccmtocystis; Chnlnrn; Ambrosiclln, in parr) systems for evolutio nary studies of a spectrum of inter­ Ophiostomatalcs actions. In o tl1cr cases tl1c interactions are of econo mic Ophiosromataccac ( Ophiostomn; Lcptogrnpbium; Ambrosiel/n, importance because they involve dispersa l of se ri o us in part; SporotbrL\~ Rnffnclcn) fungal patl1ogens o r sapstain fungi tl1at damage trees, Loculoascomycctcs- Di scom~ ' Cctcs Unknown afli nitcs-Symbiotnpbrinn crop plants, and forest products. Some of the fungi rely 13asid iomycota o n the insect fo r surviva l because they arc poor com­ 1-1y mcno mycctcs petitors with apro bcs in tl1cir habitats. The fungi that Aphyllophoralcs fo rm associati o ns include ascomycetes (yeasts, Ophios­ Corticiaccac ( E11tomocorticium and others) toma [Figs. 18.8 and 18.9], eratocystis and related Agaricalcs conidial forms, and aphyll o pho ralean basidio mycetes) Lcpiotaccac ( Cblorophyllmu, Lcucongm·icus, Tcnuitomyccs, and undescri bed forms) that are symbio nts of va ri o us g ro ups of beetl es, in trace!-

FIGURE 18.8 Ascosporcs oozing from the necr of an Ophiostomn species pcrithccium. (Ph oto by Kicr Klcpzig, DA Forest Service) - ..... r ..... ·----·-

/meet- n11d Otbcr Artbropod-Associntcd Fuugi 41 7

FIGURE 18.9 Resinous lesions on the bark of a pine tree caused by mass inoculation with OpiJiostOIIUI­ miuus, a fungal associate of the southern pine beetle. (Photo by Erich Vallery, USDA Forest Service)

species that commonly occur with phloem-feeding methods we descri be are applicable ro colonizers of hard­ beetles, usuall y in li ving trees (gro uped here as bark woods as well. N umerous fungi occupy almost all parts beetles). The other category incl udes species associated of the body surface and gut of a beetle, as well as tl1e with beetles tl1 at require fungi as a primary nutri ent tree tissue the beetle infests. Among the fungi found on reso urce in all life hi srory stages (ambrosia beetles). T he the beetl e surface and within the di gestive tract arc yeasts distinction is artificial, and the fungi often arc cl osely (Call aham and Shi fri nc 1960; Bridges et al. 1984; re lated. H owever, because techniq ues used to study the Leufvc n and Nehls 1986), various saprobcs (Bridges et phloem-feeding and ambrosial associatio ns diffe r some­ al. 1984), and ophiostomaroid fungi (sec "Identifica­ what, we discuss them separately (see "Am brosia Beetles tion," later in this chapter), especially in Ophiosto matales and Fungi" later in this chapter). We in cl ude o ur dis­ (Upadhyay 1993). Species of Ophiostoma and related cussion of phloem-feeding weevil s (Curculi o ni dae) with coni dial fungi associated with beetles include many of the bark beetles. the stain fungi known to disco lor wood (Fig. 18.11 ) Bark beetles colo ni ze both hardwood and conifer (HarringtOn 1988 and references tl1erein ). Beetle­ trees, and although we will emphasize those that colo­ associated ophiostomatalean fungi also have been impli­ nize conifers and their fungal associates, many of the cated as conifer pathogens (Harringto n and Cobb 1988; 418 Richm·d K Bc11jami11 et al.

FIGURE 18.10 Yeastlike symbiotes gathered at the epitherial plug (the junction of the ova ri ole and pedicel ) of a planrhopper. The symbiotes of pianthoppers are transmi tted to the next generation through the ovary, entering the terminal oocyte in the ovarioles posterio rl y. The symbiotes move fi·om the insect fat body to the epitherial plug when the ovary matures .

FIGURE 18.11 ross-section of a red pine infected wi tl1 blue-s tain fungi. (Photo by Kier Klcp zig, USDA Forest Service)

Harrington 1993), and certain members of this gro up Certain beetles of tl1e curculio nid subfamilies colyti­ are capable of killing trees (Brasier 1988; Harrington nae and Pl atypodinae have evolved specialized structure 1993; ol heim et al. 1993). More often, however, they kn own as myca ngia, the purpose of which appear to be are a sociated with resi nous lesio ns that may ca use the the storage, culture, and transport of fungi ( mycangia occlusion of sapwood (Harrington 1993). Some of these occur also in ambrosia beetles). The mycangia of a few fu ngi also are antagoni sts of beetles, reducing reproduc­ bark-beetle species are complex and include secretory tive succes and larval development (Barras 1970). cell s (Harrington 1993). More commonly, beetle mycan­ Altl1ough tl1e exact ecological ro les played by the va ri ous gia are less developed , simple pits in tl1 e exo keleton of ophi ostomatoid fungi have ye t to be determined, they the head, pro notum, or elytra . These imple structures are undo ubtedly closely associated witl1 bark beetles and may contain yeasts , ophiostomatalean fungi, and otl1er weevils and their tree hosts. Efforts to examine tl1 e diver- fungi, includin g corticio id basidiomycetes (Harrington ity of beetle-associated microorganisms center aro und 1993; Lewinsohn et al. 1994). M yca ngial fungi are tl1 ese fungi. tho ught to be mutuali sts of tl1 eir beetle hosts, pas ibl y

-----'='--=-----· --- - - !IIScct· nud Other Artbropod·Associntcd Fungi 419

by receiving nutrients fro m the host (Bridges 19 83; where it is left fo r sevcr3l days. The host material is Bridges and Perry 1985; Gold hammer ct al. 1990). checked dail y fo r insects moving from the soil onto the Often fungi have a yeastlike morpho logy whil e they are stem-secti on surface . In addition, some root weevils may in a mycangium, rather than the hypha! fo rm o utside the be coll ected fro m the lower stem as they ascend at night mycangium and in the environment of the wood . T he to feed o n br3nches (Kicpzig ct al. 1991). Walking raxonomy and ecology of many of these fungi arc not weevil s are fo rced in to a coll ection jar atop a screen fully known (lvloser ct al. 1995 ). funnel that is wrapped around the main stem. Bark beetles th3t attack the lower stem of trees can be coll ected in va ri o us types of flight tr3ps. A lower-stem Coll ection fli ght trap consistin g of an inverted, plastic jug modified Bark-beetle fungi can be found in or o n insects, other by having a coll ection jar 3ttached can be baited with cl1an tl1e beetles that they colo ni ze. True hosts of these turpentine and ethanol and used to collect turpentine fungi are found in only a few coleopteran fa mi li es, beetl es 3nd some root insects (Klcpzig et al. 1991 ). Tur­ including the Curculionidae, especiall y in the subfamilies pentine beetl es also can be c3pturcd in bounce traps in colytinac and Plarypodinae (Harrington 1988; Mall och whi ch the fl ying beetl e strikes a black pipe b3ited with and Blackwell 19933). Kn owledge of the host insect's ethano l and turpentine, 3nd fa ll s into a water-fi lled pool biology facilitates effective coll ection of specimens for below, fro m whi ch it is collected (Fatzinger 1985; isolation of fungi. These insects colonize tl1e lateral Phillips et al. 1988). Lindgren multiple-funnel traps roots, the root collar, the main stem , the br3nches, (Lindgren 1983) can be hung nc3r the ground for developing shoots, and even fi·uits of 3 va ri ety of trees coll ection of lower flower-stem insects fl ying tow3rd (S. L. \Vood 1982; Drooz 1985 ); tl1e best-studied attract3nts (Phillips et al. 1988). insect-fungal complexes are fou nd in coni fe rs. M3ny Most of tl1 e aggressive, tree-killing bark beetles attack bark beetles and weevils use tree- and insect-produced the central and upper portions of the stem (S. L. compounds to locate suitable hosts as well as mates (D. Wood 1982 ; D rooz 1985). Fl ight traps hung in the mid L. Wood 1982 ). Using host materi al, host compo unds, to upper cano py and baited with species-specific and/or beetle pheromones as 3ttractants during times of phero mo nes often arc used to S3mplc those insects. The easonal insect abundance, it is possible to coll ect large pro per choice and use of pheromones is, however, a com­ quantities of beetles from which fungal associates C3 n be pli cated matter. Species may respond to different com­ isolated . Adult beetles actively seeking host substratum po unds o r to the cnantiomers of those compounds (D. and/or m3tcs respond to indicators of att3ckin g beetl es L. Wood 1982 ; Payne et 31. 1984; R.bark beetle and weevil biology. overwhelmed by saprobic fungi that may interfere with Pitfall tr3ps have been used to capture root-infesting isolati o n of bcetl e-associ3tcd fungi. An alternative is to weevils and beetles within fo rests and plantatio ns (H ar­ leave tl1 e coll ectio n jar empty so that beetles arc not rington ct 31. 1985; Wircosky et al. 1987; H unt 3nd kill ed o n fa llin g in to the trap. However, bark beetle Raffa 1989). One such trap (Hunt and Raffa 1989 ) can phero mones may serve 3S b iromonal attractants for be constructed from capped sections of plastic pipe, predaceous insects, in which case the collection jars drilled witl1 small entrance holes and pbced so that the become 3 feed in g ground for predators (D. L. \Vood holes are even with the soil surface. Vials of eth3no l 3nd 1982). T he resul t is a pi le ofb3rk beetle pa rts with little turpentine arc hung inside the trap, and a secti o n of pine va lu e 3S a source of fungi. If traps 3rc sampled the S3me rem is placed in the bottom of the trap. Weevil s 3nd day they are depl oyed, however, the method C3n be used beetles cr3wl through the soil and into the entrance holes successfull y. Another 3ppro3ch is daily collection from 3 in response to the volatile materials (H unt and lbffa coll ecti o n cup cont3incd within an electric cooler to slow 1989) and are unable to escape. Stem sections (billets) predators (B.S. Lindgren, personal communication). also have been used to coll ect root-feed ing beetles 3nd !Vlany bark beetle-associated fungi can be isobtcd weevils (Lewis and Alexander 1986; T unset et al. 1988). fro m in fested host tree material. Phloem and xylem tissue The stem section usually is placed in cont3Ct with the soil fro m 3rcas aro und insect feeding sites, entrance holes, 420 R..ic/;m·d JC. Bmjnm i11 ct nl. and adult o r larval galleries ca n be coll ected aseptica ll y southern pine beetle, the pronotum is removed fl·om the for fungal isolation as can fi·ass fro m larval galle ri es head and abdomen of an adult female, and the legs are (Bridges ct a!. 1984; Harrington 1992; Solh ei m 1995). re moved from the pronotum using a pair of sterile, fine Various phoretic tarsoncmid mi tes have been impli ­ forceps. T he entire, intact pronotum is placed in sterile cated in the transmission ofbark beetle-associated fungi, distill ed water fo r 1 minute; placed in modified White' and these arthropods also can be a source of fungi fo r solution (Append ix II; Barras 1972) for 4 minutes; and cultures (Bridge and Moser 1983; Moser eta!. 1995). then passed through two ucccssive rinses with sterile Mites typically are removed from the beetl e exoskeleton d istill ed water. The pronotum then i quartered asepti­ (often underneath the beetle elytra) with fi ne needles . ca ll y with fine forceps, and the four cctions arc placed They can be cultured directly or mounted o n sli des fo r on malt extract agar (MEA) o r MEA amended with later microscopic examination of spores. 2 mg/ ml beno myl to fac ili tate the scmisclcctivc isolation o f certain pecies such as Ccmotocystiopsis rauaculoms, an isola te known as Eutomoco1·tocium species A, and pcrhap Storage other mycangial fungi (Ross ct a!. 1992). Fungi should be isolated as soon after the insects arc Beetles ca n be crushed on an agar medium to i olate collected as possible. When tl1 i is no t feas ible, insects, pho rctic fungi (Harri ngton 1992). Alternately, beetles mites, and tree tissue should be placed in steril e vials, may be ground in steril e distilled water in a glass tissue transported to the laboratory in icc-fi ll ed coolers, and ho mogeni zer. Al iquots of the homogenate arc plated refi·igeratcd until they are processed. Pl acin g small pine directl y o r d ilu ted a number of times and then plated on twigs and/ or moist paper in coll ectio n vials may increase an agar medium (Kiepzig eta!. 1991). T his technique survival of insects during periods of extended refi·igcra­ has the added advantage of all owing for quantitative esti­ tion. When possible, insects sho ul d be kept in separate mates of the number of propagulcs of each fungal species vials or containers to minimize the li kelihood of cross bein g ca rried by each insect. Sample of beetle-infested contamination between hosts. T ree-tiss ue sa mples ca n be tree tissue may be coll ected aseptically and placed directly refi·igcratcd or stored at room temperature in moist o n water agar (WA ). In many case , fungal fi·uiting struc­ chambers, which helps to keep the fungi viabl e and actu­ tures ca n be fo und lining beetle galleries; masses of all y may promote sporulation of the fungi on the host spo res ca n be transfe rred directly to media with a fine substratum ( eifcrt ct a!. 1993). Specimen ca n be d ri ed , needl e ( cifc rt ct a!. 1993). but subsequent isolation into cul ture often is not A majo r confo undi ng factor in all of the isolation tech­ succe ful. niques that we have descri bed is the pre cncc of sapro­ bi c contaminants either o n the beetle exoskeleton or within beetle-infested tree tissue. Although these fun gi Preparation of Specimens for Study may be signi fica nt components of the beetle fungal flora , tructures associated with exual or asex ual sporul ation many of them grow so quickly that they overwhelm in tl1e tree wood or bark provide diagnostic characters fo r other fungi of interest (Gibbs 1993; Seifert ct a!. 1993). identification of tl1e fungi. It is important to make good Diluting sa mples before plating often results in separa­ slides of material when it is ava il able, even if tl1 c fungus ti on of fungal colo ni es of interest that can be subcultured is to be cultured, because some of tl1 c species will not (Ki cpzig et a!. 1991). Another technique is useful fori o­ produce sexual reproductive structures in cul ture. To lating members of the Ophiostomatale , which include erve as voucher , specimen must be mounted o n mic ro­ many of the well -studied beetle associate . A character­ scope slides using the double-cover sli p metl1 od d isc ussed istic of this group of fungi is a high degree of tolerance earlier (see "Preparation of Specimens fo r Study" in the of the anti fungal compound cycloheximide ( cifcrt eta!. Laboulbcniales section for detailed protocol) and made 1993). Accordin gly, MEA, WA, or potato dextrose agar permanent. In addition, spores of fu ngi fo und in pits o r (PDA) ca n be amended with 200 ppm cycloheximide, myca ngia on beetle and mite exoskeleton ca n sometimes to inhibit growth of nonophiostomatalean fungi, and be identified using light and tran mi ssio n, and scanning­ 100 ppm streptomycin sul fate , to inhibit bacterial growth electron micro copy (Happ ct a!. 1971; Lcwinsohn ct a!. (H arrington 1992; Seife rt et a!. 1993). ome yeasts, 1994 ). however, and species of fi lamcntou fungi, including Pcnicillimn, may also grow on the c media (Harrington 1992 ). Sin gle colonies on plates may be transferred C ultivation and D eposition of Cultures (via hypha! tip transfer and/ or transfer of spore masses) Myca ngial fungi may be isolated fro m beetl es by dissect­ to unamended PDA or MEA for identificatio n of pure ing the mycangium from the beetle, surface sterili zin g the cultures. structure, and subdividing and placin g it o n selective Plates normall y are incubated at from 20- 25° , media (Barras and Perry 1972). For example, fo r the altho ugh some bark beetle associates may grow better at /meet- nnd Other Artbropod-Associntcd Fungi 421 cooler temperatures (HarringtOn 1992). Cultures ca n larvae: those that consume o nl y fungi (mycetophagous) be grown on a weak agar medium in vials and stared at and those that enlarge their gall ery or larval cradles in -20°C, or they can be lyophili zed for prese rvatio n. Serial the xylem as they develop, thus consuming both wood transfe r and stOrage on rich media generall y arc not I"C C­ and fungal material (xylo mycetophagous) . The latter ommendcd (Seifert et al. 1993). All of these fungi sho uld habit approaches the behavior of some mycangial bark be of interest tO most general culture collections because beetles. As adult beetles make brood g:1 llcries by tunnel­ of their interesting biological associations and economic ing into the new woody host materi al, they transmit importance. pecies-spccific obli gJtory fungal symbiom s in ectoder­ mal mycangia. The damage done by ambrosia beetles is caused by their boring activity and subsequent staining Preparation and Deposition of Vouchers of the wood (McLc:1n 1985). ·ouchers can be prepared by placing fungal structures The primary symbio nts of ambrosia beetle species are on plant material or cu ltures that have been dried, usu­ tl1osc that are consistently isolated from tl1e myca ngia ally in moving air at room temperature, in packets o r of adult beetles coll ected during flight, from adults mall boxes. T he specimens should have mature sexual­ excavating their new brood galleries, or from brood gal­ reproductive structures that include diagnostic characters. leri es in the prese nce of actively feeding larv:1e (Batra Permanent slide mounts arc also desirable as vouchers. 1967, 1985; Roeper et al. 1980). T hose fungi include In some cases parts of beetles and associated mites can Awbrosiella and Rn.ffaclea, asexual genera rebtcd to be mounted o r prepared for scanning electron micro­ Ophiostoma :1nd Ccratocystis. In warmer regions Fusat·­ copy to show phoretic spores of the fungi . Arthropod ium may be :1n ambrosi:1l associate. Other fungi, referred materia l can be maintained in 70% alcohol to which a to as auxiliary or secondary :1mbrosial fungi, are not little glycerine has been added. usually isola ted from myca ngia but are regularly present in the brood tunnels after beetle pup:1tion. Many of those fungi have mucilaginous spores thJt may be transmitted Identification by phoretic mites and beetles. Their presence in tunnel The fungi that occur in bark-beetle associations are a parallels that of the sapro bi c fungi associated with bark diverse lot. \Ve mentioned ophi ostomatoid fungi, which beetles and interferes with the isobtio n of the slower­ have distinct morphological features such as evanescent growin g primary symbio nts in culture. asci and long-necked ophi ostomatoid perithccia through Wood-boring scolytinc and pl atypodine ambrosia whic h the sticky ascospores arc passively discharged for beetles usuall y inhabit dead o r dying trees (see reviews arthropod di spersal. T hese species belo ng to several by Batra [1967], Francke-Grossmann [1967], Norri orders , primarily Ophiostomatalcs ( Opbiostomn.) but also [1979], Beaver [1989], :1nd Roeper [1995]). S. L. Wood Microascales ( Ceratocystis) and Pyxidiopbora. In addi­ ( 1982) described the Nortl1 and Central American tion, these orders include a number of derived asex ual Scolytinae and li sted their woody hosts and geographic fo rms such as Sporothrix, Lcptographium, and Chalara. distributions. In addition, Wood and Bri ght (1987, 1992) There arc yeasts , mycangial basidiomycetes, and other fil ­ catalogued all Scolytinae and Plarypodinac and tl1eir plant ame ntous saprobic fu ngi as well. Several helpful refer­ hosts, distribution records, and litcrawrc references. ences to these fungi are ava il able (Barras and Perry 1975; Harrington and Cobb 1988; Perry 1991; Schowalter and Collection Filip 1993; Upadhyay 1981; Wingfield ct al. 1993; S. L. Wood 1982). Ambrosia fungi are collected with their beetle associates. The fungal form is dimo rphic; in the well -developed mycangia the ambrosia fungus is yeastlike, and witl1in tl1 c AMBROSIA B EETLES AND FUNGI g:1 lleries it is filamento us but usually cropped by the feeding beetles. As we mentioned earlier, the ambrosia and bark beetles differ primarily in their nutrient sources, with adult, Beetles during Dispersal Flight. In nortl1crn tempcr­ pupal , and larval stages of the former rel yin g o n fungi, Jtc regio ns, adult beetles fly o nl y during a short period rather than phloem used by the latter. Because the sep­ (generally a month) in spring when temperatures reac h aration of these two groups is artificial, many of the l8°C (Rolin g and Kearby 1975; Turnbow and Franklin fungal genera that we discussed earlier in the sectio n 1980; Weber and McPherson 1991 ). Most species "Bark Beetles and Fungi" are discussed again here in have only a single annual fli ght, but some species (e.g., association with tl1e same beetle fa milies. H owever, the lvfonartbntm species) have two generations :1nd two interactions are different and less flexible. Roeper ( 1995) flights each yea r. In se mitropical and tropical regions, described two feeding categori es of ambrosia beetle many of the species have multiple generations but 422 Ric/;nrri K BCII}nmiu ct nl. seldom fl y during dry seasons. T he beetles tend to fl y in important fo r identification of these species bcc:1use they the late afternoon and earl y evenin g. provide JdditionJI di:1 gnostic characters such as growth Beetles can be caught li ve in fine-mesh nets, in rate Jnd pigment production. mechanical rotary traps ( R.udinsky :1nd Datcrman 1964 ), or with hand nets. Unprocessed timber at sawmills and Isolation logging operatio ns, :1nd wi nd-thrown, wind-d:1m agcd, standing supprc sed, Jnd/ or d iseased trees attract fli ghts T he pri mary fungi of an ambrosia beetle arc abundant in of ambrosia beetles. The beetles are Jttracted to ethanol a g:1 ll cry o nl~ ' when brva l stages :1re present (Kajimura produced by the fermentation of host timber. T hus and Hijli 1992). Thus, the best isolates of primary flmgal ethanol o r beer c:1n be used in additio n to timber to sy mbionts c:1n be made a month or two after initial infe - attra ct beetles to a collection site. Beetl es coll ected tJtion. G:1 ll eries Jrc exposed by sawing thin sections from should be placed individuall y into sterile vials o r stop­ the in fested bole. It is important to work as quickly and pered tubes with damp sterile fi lter p:1per and then Js JScpticall y as possible, usin g alcohol-flamed saws, cooled during tran port to the bboratory. wood chisels, :1ndj or pruning shears. Adult insects can be removed, and visibl e fungal growth within the several­ Beetles Infesting Wood. Most Jmbrosia beetles in tem­ millimeter-diameter ga ll ery ca n be isolated using sterile perate regions infest cut timber, wind-thrown trees, fi ne forceps. T hin sli ces or chi ps of gallcrie hould be pre­ wind-broken limbs o r boles of trees, Jnd/or suppressed served, dried, and mo unted, or mounted directly on slide or diseased trees. vVoody timber dead for mo re than a with fixJtive mo unting medium, such as bctophcnol­ year is seld o m infested. H owever, temperJtc-regio n anilinc blu e, for later study. ambrosia beetles of the genus Co1·thylus and warm Ambrosia fungi in the genus CortiJylus and most Xyle­ temperate-, se mitropica l-, and tropicJI-regio n members boms species generall y form a thick, whitish palisade of the scolytin e beetle tribe Xyleborini (X ylcboms and b yc r o n the w:1ll s of galleries if egg and/ or brvae are Xylosrmrlms) are capable of attacking apparently hcJithy prese nt. That fu ngal growth can be isolated easil y by and undamaged woody host . Ambro ia beetles infest strc:1kin g o r spot plating on isolation media (sec next the bole of the tree, borin g directly thro ugh the bark section o n "Culture"). into the xylem. The cntrJnce hole is se ld m more than Fungal growth usuall y is not so evid ent on the ga ll ery 1 mm in diameter; the boring fi·ass is li ght in color ini ­ w:1 ll s o r brva l cr:1dles of xylomycctoph:1gous insects; tially, but darkens as the primary symbionts begin to thus, small sli ces and chip of wood should be removed grow o r as it is contaminated with brval fecal pell ets. aseptically for plating. Sli ces o r fragments of galleries By comp:1rison, scolytinc bark beetles typica ll y produce c:1 n be placed Jscptically in a sterile moist chamber brownish boring frass bcc:1 usc they mine the inner bark (Appendix I ) to encourage fungal growth in the absence a they construct ga ll eries. of actively feeding brvac, so that primary ambrosia flmgi c:1n be isolated, often within a few day , before contam­ in ation from saprobic fungi. Storage Live beetles trapped in fli ght o r taken fi·om galleries Once infested timber is located, it sho uld be cut into arc difficult to handle because of their small size and managc:1blc lengths, generally about a meter, and smooth cylind ri ca l hapc. A simple vacuum apparatu returned to the laboratorv. The surface of the in fe sted consistin g of :1 sterile micropipette tip with a small aper­ timber is cleaned of dirt and biota :1nd disinfected by ture JttJched to :1 rubber h se fixed to a vacuum pump lightly wiping cveral time with ethano l. Once the or v:1cuum line :1 ll ows o ne to pick up individ ual beetle beetles h:1 ve bo red into the ;..:y lem, they generall y will not :1nd trJnsfe r them c:1s il y from dish to dish or to sterile reemerge unless the wood begins to dry. Painting cut glass sli des fo r dissectio n. ends with melted paraffin wax slows log dchydrJtion. Beetles can be surface disinfected to reduce the pre - If the wood is stored o ut of direct sunlight, the beetles cncc of nonmyca ngial microbes by washing in sterile will continue gallery constructio n, produce broods, :1nd 0.1% H g 12 solutio n or dilute sterile bleach (NaH 12) complete a generJtio n of their life cycles. for 2-4 minutes, followed by several rinses in sterile water. InvestigJtors ca n also free ad ult beetles of external nonmyca ngial microbe by placing them Preparation of Specimens for Study alternately in plates of sterile wet filter paper for 18 hours These methods generally are the sa me o nes used fo r and then o n dry sterile filter paper for 6 hours. bark-beetle Jssociates; however, Jmbrosia beetles may ScverJI transfers typicall y remove most ex ternal microbes. lack diagnostic character such JS sex ual stages and even Individual beetles can be stored on sterile moist filter conidia may be bcking. Consequentl y, cultures arc pbtcs for mo nths at rcfi·igcrator temperature until - -- --~~-- ..

In sect· nnd Otbcr Anbropod·A ssocinted Fnngi 423

needed for dissection and isolation. Prevention of dehy­ subisolatio n of fi lamentous fungal growth and rcstrcak­ dration appears to be the critical factor for keeping them in g of ycastlikc colo ni es usually is necessary for purifi­ alive during long-term storage. cation. Some Awbrosiella species (A. hartigii, A. fcrrugiuca, A. xylcbori, A. mlplmrca) grow rapidly in fil ­ amentous form and often produce melanin pigments. CuJnn·e Awbrosiclla bnmuea, A. guat!Jotrichi, and most R.affac­ Primary ambrosia fungi are abundant in mycangia at the lca species fo rm ycastlikc mycel ial colonies initially and time of Aight and/ or early stages of gall ery devel opment sho uld be subcultured by streaking and hypha! tipping. (Roeper 1988; Kajim ura and Hijli 1992). For that reason Yeasts commonly are encountered and should be sub­ timi ng is important for isolation of the true primary sym­ cul tured by streaking. biotic fungi from a beetle. T he sex of the beetle also is Several culture media, including PDA, malt important because myca ngia usuall y develop o nl y in the extract-yeast ex tract agar (MEYE), and dilute malt ex that initiates the brood gall ery system. T he beetles extract-yeast extract-glu cose agar (sec Appendix II ), can usually have a single pair of myca ngia, whose positions be used to culture primary mycangial sy mbionts and can vary even between ve ry closely related beetles. isolate fim gi fro m gall eries and mycangia of a particular tvlycangia of scolytine beetles can be o ral , pro no ta l, beetle. As many isolations as possible should be mesonotal, prothoracic pleural, promesonotal, o r clytral; attempted from available collected material. Frequency in plat:ypodincs tl1e mycangia are pitlike. If the in vesti­ of occurrence of a particul ar microbe sho ul d establish the gator docs not know where myca ngia are located, he o r presence o f associated symbiont microbes. Prokaryotic he must examine all the beetle's body parts carefu ll y micro bes are seldo m encountered, so antibiotics gener­ under a microscope. all y arc not used in isolatio n media. Once axenic cultures Mycangia are dissected ft·o m the beetle, and the con­ have been made, they can be stored on slants of dilute tents should be plated onto agar medium for isolatio n. malt extract-yeast cxtract-gl ucosc agar for fu tu rc study. The beetle should be dissected under a dissecting microscope on steril e alco hol-flamed glass slides in three separate drops of sterile sa line o r bovine serum usin g Preparation and Deposition of Vouchers alcohol-flamed fine watchmaker forceps, fin e needles, Procedures fo r preparation and deposition of vouch­ and sterile micropipettes. T he body parr o f the beetle ers arc similar to those for bark-beetle associates; containin g the mycangium(a) sho uld be separated from however, these fungi do not produce sexual states. It is the rest of tl1e body in the first of the three drops. In important to prepare vouchers from ea rl y cultures the second drop, the mycangium(a) is separated. T he because ambrosia fungi may stop producing conidia after mycangium( a) is broken apart in the third drop and a few transfers. plated. The presence of fungal cell s can be ve rified using the low power of a compound microscope. Myca ngial fungal cells sink to tl1e bottom of the drop, whereas Identificatio n insect fat droplets, with which they may be confused , stay in suspension. The mycangial fungi tend to be ycastlikc Primary fungi (Ambrosiclla and Raffaelea species) can be budding forms or, if the fu ngi are proliferating ac ti vely identified usin g the works of Batra ( 1967) and Roeper in the mycangium, monili o id chain s. All parts of the and coll eagues (1980). Identification ofthe beetle asso­ mycangia arc spot plated and/or streaked o n isolation ciate is impo rtant because the fungi are usuall y host media (see following pa ra graphs) and incubated at specific. Many of the fi lamentous ambrosia fungi t-ail 22~25 ° C. Sterile micropipettcs ca n be used to pick up to sporulate in culture or after repeated subculture. small masses of fungal materi al to be plated. H owever, increasi ng ni trogen content (L-proline) of the Prccoxal mycangia (e.g., in species of 1\.-fonarthrmn medium, buffering the agar to a pH ncar neutral, and

and Gnatbotriclms) can be d issected to remove their elevatin g C0 2 levels in the culture container may induce fungi, or the mycangia can be sampled directly. The adult sporulatio n in the fungi. Molecular studies have been beetle is killed and fixed ve ntral side up on a sterile glass used to characteri ze species of Ambrosiclla and Raffae­ slide with a drop of molten paraffin. U nder a dissecting lca and have shown that Awbrosiella is polyphyletic with microscope, the forelegs arc removed, and then a sharp some species being related to Cemtocystis and others needle is used to remove the contents of tl1 e enlarged alli ed with Ophiostoma (Cassar and Blackwell 1996). A coxal mycangium; that materi al is plated directly o nto similar study showed that Raffaelca species arc related isolation medium. to Ophiostoma (Jones and Blackwell 1998). Ref-e rence Plates should be examined daily after isolations have cultures of most known primary ambrosia fungi arc been made from a gallery o r mycangium. H yphal-tip ava il able from ATCC and CBS.