Seasonal Dispersal of the Oak Wilt Fungus by Colopterus truncatus and Carpophilus sayi in Minnesota

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1 Sesonl Dispersl of the Ok Wilt Fungus by Colopterus trunctus nd Crpophilus syi in Minnesot Angie K. Ambourn, Deprtment of Entomology, University of Minnesot, St. Pul 55108; Jennifer Juzwik, USDA Forest Service, North Centrl Reserch Sttion, St. Pul, MN 55108; nd Roger D. Moon, Deprtment of Entomology, University of Minnesot, St. Pul ABSTRACT Ambourn, A. K., Juzwik, J., nd Moon, R. D Sesonl dispersl of the ok wilt fungus by Colopterus trunctus nd Crpophilus syi in Minnesot. Plnt Dis. 89: Sp beetles (Nitidulide) re considered importnt overlnd vectors of the ok wilt pthogen, Certocystis fgcerum, in the north centrl United Sttes. Colopterus trunctus nd Crpophilus syi re thought to be the principl sp beetle vectors in Minnesot. Field studies using windoriented funnel trps bited with ggregtion pheromones of the insects were conducted during 2 yers in est centrl Minnesot. The studies compred temporl flight dynmics of Colopterus trunctus nd Crpophilus syi from April through October, mesured the proportion of dispersing dults of ech species crrying vible pthogen propgules, nd estimted the popultions of contminted dispersing beetles in ok (Quercus spp.) stnds with nd without ok wilt. Abundnce of Colopterus trunctus peked in either April or My, but the pthogen ws most frequently isolted (20 to 45%) from beetles obtined from ok wilt sites from July through September. However, the highest contminted insect popultion (CIP) generlly occurred during April nd My. Abundnce of Crpophilus syi peked in October of both yers, but the pthogen ws most commonly isolted from beetles collected during My nd June. The CIP ws highest in June. The infective insect potentil, s indexed by CIP, ws gretest for both species in the spring nd ws greter for Colopterus trunctus thn for Crpophilus syi. Additionl keywords: fungl phoresy rtes, vsculr wilt disese Ok wilt, cused by Certocystis fgcerum (Bretz) Hunt, is spred overlnd by insect vectors such s sp beetles (Coleopter: Nitidulide) (15). Insect trnsmission is significnt becuse it is the mens by which new ok wilt infection centers re estblished. Sp beetles trnsmit infectious propgules from the pthogen s sporulting mts formed on recently killed oks (Quercus spp.) to fresh wounds on helthy oks (8,16,21,23,26). Mny species of sp beetles hve been ssocited with the ok wilt disese cycle. Bsed on his review of the literture, Sklbeck (27) reported 27 different species Corresponding uthor: J. Juzwik E-mil: jjuzwik@fs.fed.us Current ddress of A. Ambourn: USDA Forest Service, Forest Helth Protection, 3700 Airport Wy, Firbnks, AK Mention of trdemrk, proprietry product, or vendor does not constitute gurntee or wrrnty of the product by the USDA, nd does not imply its pprovl to the exclusion of other products or vendors tht my lso be suitble. Accepted for publiction 6 June DOI: / PD This rticle is in the public domin nd not copyrightble. It my be freely reprinted with customry crediting of the source. The Americn Phytopthologicl Society, ssocited with ok wilt fungl mts nd 38 different species ttrcted to wounds on oks. However, little distinction hs been mde s to the reltive importnce of species-specific vectors in pthogen trnsmission (25). Norris (26), however, suggested tht some species might hve greter ecologicl speciliztion in their ssocition with the pthogen thn others. Sp beetle species dispersing in ok wilt centers, the species ssocited with fungl mts, nd the frequency with which ech crried vible propgules of the pthogen were studied in Minnesot in the erly 1980s (18). Glischrochilus qudrisigntus Olivier, G. fscitus Sy, G. snguinolentus Olivier, nd G. siepmnni Brown were the most commonly cptured free-flying nitidulid species (in descending order of frequency) trpped in dough- nd melonbited trps. Colopterus trunctus Rndll ws the most common species present in ok wilt mts, nd Glischrochilus, Epure, Colopterus, nd Crpophilus species were contminted with C. fgcerum (17). Further studies were conducted in Minnesot to test the hypothesis tht ll sp beetles tht hve been implicted s C. fgcerum vectors re importnt in the pthogen s trnsmission in nture (5). Colopterus trunctus nd Crpophilus syi Prsons were found to ccount for 54% of the 2,542 nitidulid beetles collected from fungl mts. Fungl isoltions reveled these two species lso were crrying high numbers of vible propgules (>3,000 CFU per beetle) (5). Colopterus trunctus nd Crpophilus syi lso were found to ccount for 95% of ll sp beetles ttrcted to fresh wounds in subsequent study (20,21). Furthermore, 78% of the beetles were contminted with the pthogen. Thus, these two species re currently considered the principl sp beetle vectors of C. fgcerum during spring in Minnesot. Studies were undertken in 2002 to lern more bout the temporl flight dynmics of Colopterus trunctus nd Crpophilus syi throughout the growing seson in Minnesot. Such knowledge could led to new control methods for overlnd trnsmission or led to refinements of existing guidelines for disese prevention. New light lso my be shed on the reltive importnce of ech species in spreding the pthogen. New quntittive informtion could be useful in developing models to predict sesonl risks ssocited with the infective insect potentil for C. fgcerum by specific insects. In order to ccomplish this, the time of yer when C. fgcerum contminted beetles re dispersing in ok wilt stnds must be identified, s well s ny differences in vectoring potentil tht my exist between the species. Temporl flight dynmics of Colopterus trunctus hve been studied in Minnesot to some extent. Pek bundnce of this species hs been shown to occur in erly spring (22,27). There hs been some chrcteriztion of Crpophilus syi flight ctivity in Wisconsin (23); however, temporl flight dynmics for Crpophilus syi in Minnesot hve not been determined. Difference in occurrences of Colopterus trunctus nd Crpophilus syi in wounds nd mts hs been documented. In Iow, Norris (26) showed Colopterus trunctus rrives in the mts erlier thn Crpophilus syi nd colonizes younger mts. Colopterus trunctus lso ws more bundnt thn Crpophilus syi in infectble wounds. Field studies were initited in est centrl Minnesot to compre nd contrst Colopterus trunctus nd Crpophilus syi in reltion to the ok wilt disese cycle. Specific objectives were to: (i) determine the bundnce of dispersing Colopterus trunctus nd Crpophilus syi over the Plnt Disese / October

2 growing seson in ok stnds with nd without ok wilt, (ii) determine the proportion of the dispersing beetles of ech species contminted with C. fgcerum, nd (iii) estimte the contminted beetle popultions in smpled stnds s mesure of the infective insect potentil of ech species during spring, summer, nd fll. MATERIALS AND METHODS Study sites. Beetle popultions were studied t six ok stnds ner the Minnepolis/St. Pul, MN, metropolitn re in 2002 nd Selected stnds included ok wilt sites with colescing infection centers t Crlos Avery Wildlife Mngement Are, Wshington County; nd privtely owned woodlot in Columbus Township, Anok County. Non-ok wilt sites included privtely owned woodlot in Lke Elmo, Wshington County; nd privtely owned woodlot in Pine Springs, Wshington County. Two dditionl sites (one ok wilt nd one non-ok wilt) were studied in 2003 in the southern prt of the Minnepolis/St. Pul metropolitn re t Murphy-Hnrehn Prk, Scott County. Abundnce of beetles. Crpophilus syi nd Colopterus trunctus were cptured with wind-oriented funnel trps (10). Trps were plced pproximtely 1.5 m bove the ground nd 10 m prt. The common collection vil on ech trp contined loosely pcked, shredded pper intended to reduce the possibility of crosscontmintion (of pthogen propgules) mong cptured insects. Bits used for the Colopterus trunctus trps in 2002 nd 2003 consisted of high dose (3.5, 500, nd 280 µg of components 1, 2, nd 3, respectively) of the Colopterus trunctus pheromone (Gret Lkes IPM, Vestburg, MI) nd 10 ml of viscous, fermenting whole whet bred dough (22). Two Colopterus trunctus trps were plced t ech site. Bits used for the Crpophilus syi trps in 2002 were ccording to Brtelt et l. (4), where the species ws cptured in four different types of trps. These included high-dose (55 nd 450 µg of components 3 nd 4, respectively) Crpophilus syi pheromone (Gret Lkes IPM) nd 10 ml of viscous, fermenting bred dough; low-dose (6 nd 45 µg of components 3 nd 4, respectively) Crpophilus syi pheromone plus bred dough; high-dose Crpophilus syi pheromone only; nd low-dose Crpophilus syi pheromone only. Four trps (one of ech type) were plced t ech site in Bits used for the Crpophilus syi trps in 2003 consisted of high dose (55 nd 450 µg of components 3 nd 4, respectively) of Crpophilus syi pheromone (Gret Lkes IPM) nd 10 ml of viscous, fermenting bred dough. Two such trps were plced t ech site in Trps were emptied nd re-bited weekly from Tble 1. Best-fitting models resulting from Poisson regression nlyses of significnt explntory vribles tht ffect men numbers of Colopterus trunctus dults cught in wind-oriented funnel trps in ok wilt nd non-ok wilt sites in est centrl Minnesot from April to October 2002 nd 2003 Yer Vrible Level Estimte SE Wld χ 2 P vlue 2002 Constnt < Month My < June < July < August < September < October < Site type Non-ok wilt < Sex Femle >0.07 Interction My*site < June*site < July*site <0.002 August*site >0.80 September*site >0.80 October*site > Constnt < Month My < June < July < August >0.10 September < October <0.002 Site type Non-ok wilt <0.03 Sex Femle < Interction My*site <0.03 June*site >0.10 July*site >0.08 August*site < September*site >0.90 October*site >0.10 Fitted model for: Monthly men no. beetles cught = constnt + month + site + sex + month*site. Results of contrsts between levels of the coefficients nd the first level (implied to be 0) re presented. 11 April to 18 October 2002, nd 10 April to 16 October Beetle processing. Adult beetles were removed from trps, identified, their sex determined, nd stored individully in sterilized, micro-centrifuge tubes t 2 C until further processed. Representtive individuls of ech species were deposited in the Insect Museum, Deprtment of Entomology, University of Minnesot, St. Pul, by A. Ambourn. Within 6 months of cpture, 0.5 ml of wter ws dded to ech ded specimen nd homogenized for 5 s using n ultrsonic tip sonictor to dislodge fungl propgules on the exoskeleton nd in the gut. The resulting solution ws then serilly diluted (10-, 100-, nd 1,000-fold), nd 0.5-ml liquots of ech dilution were spred onto triplicte lctic cid-mended potto dextrose gr (PDA) pltes, for totl of nine pltes per beetle. The number of C. fgcerum colonies ws recorded 10 dys fter the pltes hd been incubted t 24 C in the drk (5). Colony ppernce nd sexul spore chrcteristics were used to identify C. fgcerum (3). Sttisticl nlyses. Counts of Colopterus trunctus nd Crpophilus syi obtined in the trps were nlyzed using Poisson regression in ARC (6). Results from ech yer were nlyzed seprtely becuse vrinces were different between yers. For ech yer, counts of beetles cught in wind-oriented funnel trps were the response vribles, nd site type (ok wilt versus non-ok wilt), month, sex, nd ll two-wy interctions were the predictor vribles. The most prsimonious model ws obtined through bckwrd elimintion, which sequentilly removed inconsequentil terms bsed on the Mllows C I sttistic (6). The significnce of ech remining term ws interpreted using the Wld χ 2 test. Fitted men beetle counts per site for ech species nd sex by month nd site type were obtined from the Poisson regression nlysis tble dt function. These vlues were used for grphing beetle temporl flight dynmics over the course of the study. Stndrd errors for the fit mens were clculted s SE = becuse in Poisson distribution the men is ssumed to be equl to the vrince (6). Presence of C. fgcerum propgules on, or in, Colopterus trunctus nd Crpophilus syi ws nlyzed using binomil regression (6). Ech specimen ws scored s 0 if propgules were bsent, or 1 if present. Results for ech species nd yer were nlyzed seprtely, with initil model terms for site, month, sex, nd ll two-wy interctions. Agin, bckwrd elimintion ws used to obtin the most prsimonious model (6). x n 1068 Plnt Disese / Vol

3 The proportion of C. fgcerum contminted beetles by species nd sex for ech smpling month nd site type ws determined by dividing the number of beetles testing positive for C. fgcerum by the totl number of beetles tested. The stndrd error for the proportion contminted ws clculted using SE = p ( 1 p) n s per Esker nd Nutter (12), where p is the proportion of smples positive, 1 p is the proportion of smples negtive, nd n is the totl number of Crpophilus syi or Colopterus trunctus tested. To combine trp ctches with levels of contmintion, the contminted insect popultion (CIP) ws clculted for mle nd femle Colopterus trunctus nd Crpophilus syi for ech month nd yer. CIP is n estimtion of the popultion of presumbly infective insects (i.e., those hving the bility to effect the trnsmission of pthogen), nd ws indexed by the product of the fitted monthly beetle ctches per site nd the corresponding proportions tht were contminted (12). The stndrd error for CIP ws clculted ccording to Freese (13). RESULTS Sesonl bundnce of Colopterus trunctus. In 2002, results of Poisson regression indicted tht numbers of cptured beetles vried cross months (P < ) nd by site type (P < ) (Tble 1). Beetle ctches were highest in April (Fig. 1). Ctches in ok wilt sites were greter thn in non-ok wilt sites (Tble 1). Overll, bundnce trends in both site types decresed s the seson progressed (Fig. 2A nd B), but they differed slightly, s indicted by the difference in site month interction for April through July (P < 0.01, Tble 1). There ws no difference in men number of mles nd femles trpped; terms for sex nd ll other twowy interctions were not significnt (P > 0.1). Abundnce of Colopterus trunctus in 2002 exhibited unimodl pttern (Fig. 2A nd B). Pek flight occurred in the week of 18 April, when 599 insects were trpped. Over the entire yer, 97% (N = 1,461) of the specimens were trpped between 18 April nd 30 June. The sex rtio ws 1.0:1.1 femles to mles (N = 1,461). In 2003, results of Poisson regression indicted tht ctches vried by month (P < , except August, P > 0.1), cross sites (P < 0.03), nd by sex (P < ) (Tble 1). Ctches in ok wilt sites were greter thn in non-ok wilt sites (Fig. 1). Ctches of mles were greter thn of femles, except in ok wilt sites in August. Overll bundnce incresed shrply in My (Fig. 2C nd D), decresed, nd then leveled off, except in August nd October, s indicted by the site month interction. All other two-wy interctions were not significnt (P > 0.1). Ptterns of bundnce in 2003 were different from tht observed in 2002 (Fig. 2C nd D). Pek flight occurred in the week of 1 My, when 356 insects were cught. Over the entire yer, 74% (N = 1,814) of ll specimens were cught between 17 April nd 26 June. In 2003, there ws second pek in the week of 21 August (N = 86) nd third pek in the week of 9 October (N = 76). The sex rtio ws 1.0:1.5 femles to mles (N = 1,814). Sesonl bundnce of Crpophilus syi. In 2002, results of Poisson regression indicted tht number of beetles cptured vried cross months (P < 0.002) nd by site type (P < ) (Tble 2). Ctches of the beetles were lowest in April (Fig. 1). Ctches were greter in non-ok wilt sites thn in ok wilt sites (Tble 2). Overll, bundnce incresed from April to July, decresed in August nd September, nd incresed shrply in October (Fig. 3A nd B). Sex nd ll two-wy interctions were not significnt (P > 0.07). Pek flight oc- Fig. 1. Men numbers of Colopterus trunctus (A nd B) nd Crpophilus syi (C nd D) cptured ech week in four sites, by yer nd site type. Insects were cught in wind-oriented funnel trps using species-specific pheromones from April to October in est centrl Minnesot. Plnt Disese / October

4 curred in the week of 4 October, when 68 insects were cptured (Fig. 1). Two other very smll peks occurred in the week of 20 My (N = 20) nd in the week of 18 July (N = 36). Over the entire seson, 58% (N = 320) of the specimens were cptured between 16 My nd 1 August. The sex rtio ws 1.0:1.0 femles to mles (N = 319). In 2003, results of Poisson regression indicted tht ctches were greter in nonok wilt sites thn in ok wilt sites (P < ) (Tble 2). Overll bundnce incresed s the seson progressed (Fig. 1, Fig. 3C nd D). Terms for month, sex, nd ll two-wy interctions were not significnt (P > 0.1). Ptterns of bundnce in 2003 were similr to those observed in 2002, but more insects were cught overll. The highest number of beetles (N = 214) ws cptured during the week of 9 October (Fig. 1). Interestingly, 163 of the specimens were cught in one trp. Three other very smll peks were found the weeks beginning 22 My (N = 83), 24 July (N = 51), nd 18 September (N = 88). Over the entire seson, 44% (N = 887) of ll specimens were cught between 1 My nd 7 August. The sex rtio ws 1.1:1 (N = 887). Contmintion by C. fgcerum. In 2002, results of binomil regressions indicted the presence of C. fgcerum on Colopterus trunctus vried cross months (P < 0.03, except July nd October, P > 0.8), by site type (P < 0.002), nd by sex (P < 0.006) (Tble 3). My, June, August, nd September hd higher proportions of individuls with C. fgcerum propgules thn were present in April. The proportion of individuls contminted with C. fgcerum in ok wilt sites ws greter thn in non-ok wilt sites. Finlly, higher proportion of mles were crrying C. fgcerum propgules thn femles. No two-wy interctions were significnt (P > 0.8). The proportion of Colopterus trunctus crrying vible propgules of C. fgcerum in 2002 rnged from 0.00 to 0.63 (Tble 4). For beetles yielding C. fgcerum, the verge CFU per beetle rnged from 6 to 1,800 (Tble 4). The proportions of Colopterus trunctus crrying C. fgcerum in 2003 vried cross sites (P < ) (Tble 3). Greter proportions of individuls were crrying C. fgcerum propgules in the ok wilt sites thn in non-ok wilt sites (P < ). Terms for month, sex, nd twowy interctions were not significnt (P > 0.1). The proportion of Colopterus trunctus crrying C. fgcerum in 2003 rnged from 0.07 to 0.46 (Tble 4). For beetles yielding C. fgcerum, the verge CFU per beetle rnged from 55 to 28,000. Results of binomil regressions for 2002 dt indicted contmintion rtes for Fig. 2. Fitted men counts per site ± SE for Colopterus trunctus mles (closed circle/solid line) nd femles (open circle/dshed line) cptured per month in four sites, by yer nd site type (ok wilt, A, 2002 nd C, 2003, or non-ok wilt, B, 2002 nd D, 2003). Insects were cught in wind-oriented funnel trps using species-specific pheromones from April to October in est centrl Minnesot Plnt Disese / Vol

5 Tble 2. Best-fitting models resulting from Poisson regression nlyses of significnt explntory vribles tht ffect men numbers of Crpophilus syi dults cught in wind-oriented funnel trps in ok wilt nd non-ok wilt sites in est centrl Minnesot from April to October 2002 nd 2003 Yer Vrible Level Estimte SE Wld χ 2 P vlue 2002 Constnt < Month My < June < July < August < September < October < Site type Non-ok wilt < Constnt >0.70 Month My >0.70 June >0.70 July >0.70 August >0.70 September >0.70 October >0.70 Site type Non-ok wilt < Fitted model for: Monthly men no. beetles cught = constnt + month + site + sex + month*site. Results of contrsts between levels of the coefficients nd the first level (implied to be 0) re presented. Crpophilus syi did not differ mong months (P > 0.9), by site type (P > 0.9), or by sex (P > 0.6) (Tble 5). The proportion of Crpophilus syi crrying vible propgules of C. fgcerum in 2002 rnged from 0.06 to 0.25 (Tble 6). For beetles yielding C. fgcerum, the verge CFU per beetle rnged from 21 to 17,000. In 2003, results of binomil regression indicted contmintion levels on Crpophilus syi vried cross months (P < 0.02, except August, P > 0.8), mong sites (P < ), nd between sexes (P < 0.03) (Tble 5). The proportion of individuls crrying C. fgcerum in ll months except June ws less thn in My. The proportion contminted ws greter in ok wilt sites thn in non-ok wilt sites, nd more femles were crrying fungl propgules thn mles. No two-wy interctions were significnt (P > 0.1). The proportion of beetles contminted with C. fgcerum in 2003 rnged from 0 to Fig. 3. Fitted men counts per site ± SE for Crpophilus syi, sexes combined, cptured per month in four sites, by yer nd site (ok wilt, A, 2002 nd C, 2003, or non-ok wilt, B, 2002 nd D, 2003). Insects were cught in wind-oriented funnel trps using species-specific pheromones from April to October in est centrl Minnesot. Plnt Disese / October

6 0.833 (Tble 6). For beetles yielding C. fgcerum, the verge CFU per beetle rnged from 9 to 43,000. Contminted insect popultions. Contminted insect popultions (CIP) for Colopterus trunctus in 2002 were gretest in April, My, nd/or June in both site types (Fig. 4A nd B). Pek CIP occurred in ok wilt sites in April (pproximtely 0.6 infective beetles per trp). Colopterus trunctus mles represented higher CIP thn femles in the ok wilt sites in April nd My (Fig. 4A). In the non-ok wilt sites, there ws little difference between mles nd femles (Fig. 4B). In 2003, mle nd femle Colopterus trunctus CIP ws similr to levels observed in 2002 (Fig. 4C nd D). Pek CIP occurred in the ok wilt sites in My (pproximtely 1.3 infective beetles per trp), nd the overll CIP decresed s the seson progressed. Contminted insect popultions were highest in My nd June, then decresed nd leveled off through September nd October when slight pek occurred (Fig. 4D). In both site types, the CIPs for mles were slightly greter thn for femles. Contminted insect popultions for Crpophilus syi in 2002 were very low (Fig. 5A nd B). Pek CIP occurred in the non-ok wilt sites in June (pproximtely 0.08 infective beetles per trp) (Fig. 5B). In generl, CIP decresed fter June nd then incresed gin in October. Overll, the CIP of femles ws slightly higher thn for mles in both ok wilt nd non-ok wilt sites. Tble 3. Best-fitting models resulting from binomil regression nlyses of significnt explntory vribles tht ffect presence of Certocystis fgcerum propgules on Colopterus trunctus dults cught in wind-oriented funnel trps in ok wilt nd non-ok wilt sites in est centrl Minnesot from April to October 2002 nd 2003 Yer Vrible Level Estimte SE Wld χ 2 P vlue 002 Constnt < Month My < June < July >0.80 August <0.03 September < October >0.90 Site type Non-ok wilt <0.002 Sex Mles < Constnt >0.80 Month My >0.80 June >0.80 July >0.80 August >0.90 September >0.90 October >0.80 Site type Non-ok wilt < Sex Mles >0.10 Fitted model for: Probbility of isolting fungus = constnt + month + site + sex. Results of contrsts between levels of the coefficients nd the first level (implied to be 0) re presented. In 2003, the CIPs for Crpophilus syi were different thn in 2002 (Fig. 5C nd D). Pek CIP in ok wilt nd non-ok wilt sites occurred in June (pproximtely 0.16 nd 0.07 infective beetles per trp, respectively). Overll, CIP incresed until June, declined through July nd August, nd incresed gin in September nd October in ok wilt sites. In generl, CIP of mles ws higher in the spring thn for femles, but dropped off shrply s the seson progressed. Contminted insect popultion of femles ws higher thn of mles in July, September, nd October. For both Colopterus trunctus nd Crpophilus syi, CIP estimtions of zero were either due to no beetles being trpped in given month (s in September 2002 for Colopterus trunctus, Fig. 3A nd B) or to the fct tht no beetles yielded C. fgcerum upon isoltion (s in ll other cses). DISCUSSION Highest numbers of Colopterus trunctus dispersing in ok stnds occurred in erly to mid-spring of both yers, with numbers decresing s the growing seson progressed. This time of pek bundnce coincided with the historicl criticl spring period for overlnd trnsmission of C. fgcerum to helthy oks in Minnesot (14). The pek lso ws consistent with results of Kyhl et l. (22) in which 98% of similrly trpped Colopterus trunctus were found between mid-april nd mid-june. A mjor spring flight pek followed by smller fll pek s ws seen in this study ws reported for Colopterus trunctus in severl previous studies in Illinois, Minnesot, nd Wisconsin (7,11,19,23,27). In contrst, the highest numbers of Crpophilus syi were cught in October of both yers. In generl, bundnce of this Tble 4. Certocystis fgcerum (Cf) contmintion proportions nd CFU from mle nd femle dult Colopterus trunctus resulting from seril dilution plting ssys in 2002 nd 2003 Yer Site type Month 1072 Plnt Disese / Vol ssyed positive Mles Proportion with Cf Men CFU/ beetle ± SE ssyed positive Femles Proportion with Cf Men CFU/ beetle ± SE 2002 Ok wilt b April ± c My ± ± 510 June ± ± 230 August ± ± 250 September ,800 ± Non-ok wilt d My ± ± 5 June ± ± Ok wilt e My ,400 ± 1, ,700 ± 2,500 June ,000 ± 19, ± 97 July ,300 ± 1, ± 730 Non-ok wilt f My ,800 ± 1, ,300 ± 2,100 June ± ± 160 October ± CFU of C. fgcerum counted on potto dextrose gr following the 10-dy incubtion period fter seril dilution plting. b In July, 5 mles nd 4 femles were ssyed; none were positive. In October, 2 mles nd 1 femle were ssyed; none were positive. c Clcultion not pplicble. d In April, 28 mles nd 25 femles were ssyed; none were positive. July to October, only 7 mles were ssyed; none were positive. e In April, 71 mles nd 39 femles were ssyed; none were positive. August to October, 35 mles nd 28 femles were ssyed; none were positive. f In April, 47 mles nd 20 femles were ssyed; none were positive. July to September, 25 mles nd 13 femles were ssyed; none were positive.

7 species incresed s the growing seson progressed, with severl distinct peks occurring during the summer months. This is the first published dt on Crpophilus syi bsed on dults cught in pheromonebited flight trps. Flight periods were previously reported for Crpophilus syi cught in bnn-bited trps in Wisconsin (23). In tht study, the highest numbers of beetles were cught in My, lte July, nd mid-september. Differences in biology, life history, nd temporl flight dynmics of these two species my be responsible for vrying numbers of beetles in the bited flight trps. Sklbeck (27) showed progressive rrivl of nitidulid species in fungus-bited trps over the growing seson in Minnesot. Colopterus trunctus ws one of the first species present in the spring, wheres Crpophilus species did not pper until week or more lter. Sklbeck (27) suggested tht differences in cpture rtes of the two species of beetles t given site my be ffected by differences in type of overwintering site, developmentl stge tht overwinters, nd the mount of het tht is bsorbed by developmentl substrte of overwintering sites in the spring. The bundnce of Colopterus trunctus nd Crpophilus syi in ok wilt ffected stnds cn be mesured in t lest three different situtions: those dispersing through the stnd, those in fresh wounds on helthy trees, nd those in ok wilt fungl mts. In this study, dispersing beetles were mesured using pheromone food synergist bit trps not locted on live or ded ok trees. These cptured beetles my hve come from outside the stnd, or my hve deprted recently from ging ok wilt mts or wounds on trees in the stnd. The rrivl nd deprture of beetles from fresh wounds nd mts hve been documented Tble 5. Best-fitting models resulting from binomil regression nlyses of significnt explntory vribles tht ffect presence of Certocystis fgcerum propgules on Crpophilus syi dults cught in wind-oriented funnel trps in ok wilt nd non-ok wilt sites in est centrl Minnesot from April to October 2002 nd 2003 Yer Vrible Level Estimte SE Wld χ 2 P vlue 2002 Constnt >0.90 Month My >0.90 June >0.90 July >0.90 August >0.90 September >0.90 October >0.90 Site type Non-ok wilt >0.90 Sex Mles > Constnt <0.003 Month June <0.001 July <0.02 August >0.80 September < October < Site type Non-ok wilt < Sex Mles <0.03 Fitted model for: Probbility of isolting fungus = constnt + month + site + sex. Results of contrsts between levels of the coefficients nd the first level (implied to be 0) re presented. (5,21,26). Cese nd Juzwik (5) reported Colopterus trunctus s the most bundnt species on immture nd mture mts in mid- to lte My, but the species presence ws much reduced on ging mts during the sme time period. Crpophilus syi ws present only in low numbers on the immture nd mture mts, but ccounted for pproximtely one-third of the totl nitidulid beetles on ging nd declining mts. J. F. Kyhl (unpublished) lso found differences in dult nitidulid beetles species nd their bundnces on mts between spring nd fll. Temporl differences in sp beetle species occurrence lso hve been reported for wounds. In West Virgini, Dorsey nd Lech (9) found the highest numbers of sp beetle species in wounds in My nd erly June; none were found in wounds in July. Norris (26) found Colopterus trunctus ws most bundnt in <2-week-old wounds in Iow during April, My, nd June; Crpophilus syi lso ws present during this sme time period. In Minnesot spring wounding study, >95% of sp beetles collected from <6-dy-old wounds were Colopterus trunctus nd Crpophilus syi (21). Very few beetles were collected in April nd June; the mjority were collected in My. McMullen et l. (24) found both Colopterus trunctus nd Crpophilus syi common in 1-week-old xe blze wounds on oks from the end of My through June in Wisconsin. For beetles collected during spring in this study, the proportions of beetles contminted with C. fgcerum were the highest in June. However, the highest contmintion rtes for Colopterus trunctus were found for the September (45%) nd July (33%) collections. The pthogen lso ws isolted from Crpophilus syi trpped in July, September, nd October, but t low rte (<5%). The high isoltion frequency for Colopterus trunctus trpped during summer months could be Tble 6. Certocystis fgcerum (Cf) contmintion proportions nd CFU from mle nd femle dult Crpophilus syi resulting from seril dilution plting ssys in 2002 nd 2003 Yer Site type Month ssyed positive Mles Proportion with Cf Men CFU/ beetle ± SE ssyed positive Femles Proportion with Cf Men CFU/ beetle ± SE 2002 Ok wilt b June c ± 66 October ± 21 Non-ok wilt d June ,000 ± 10, ,300 ± 5, Ok wilt e My ± ± 250 June ,700 ± 2, ,000 ± 4,000 July ,000 ± 4,000 September ± 18 October ± 8 Non-ok wilt f My ± ± 33 June ,300 ± 1,100 CFU of C. fgcerum counted on potto dextrose gr following the 10-dy incubtion period fter seril dilution plting. b No beetles were ssyed in April or My. July to September, 7 mles nd 13 femles were ssyed; none were positive. c Clcultion not pplicble. d In April nd My, 13 mles nd 24 femles were ssyed; none were positive. July to October, 101 mles nd 86 femles were ssyed; none were positive. e In April, no beetles were ssyed. In August, 12 mles nd 13 femles were ssyed; none were positive. f In April, no beetles were ssyed. July to October, 210 mles nd 210 femles were ssyed; none were positive. Plnt Disese / October

8 misleding, however, becuse of the reltively low numbers of dults collected (N = 41, July to September) compred with the spring months (N = 2,115, April to June). If one ssumes tht the pheromone bits for Crpophilus syi nd Colopterus trunctus re eqully efficient in ttrcting beetles of the sme species, the thret or infective insect potentil posed by these C. fgcerum contminted beetles to helthy oks over the growing seson is perhps better expressed by the CIP vlue. Proportions of contminted beetles differed between sites with ok wilt nd those without, nd is likely ttributble to the presence or bsence of ok wilt mts (i.e., sites of propgule cquisition by beetles) in the smpled stnds. It is interesting tht even when sp beetle dults were collected directly from ok wilt mts, the frequency of isoltion of the pthogen from the beetles ws not necessrily 100%. For exmple, of 57 Colopterus trunctus collected from mts between 1 April nd 27 My, 94% were contminted, while only 33% of mt-inhbiting Crpophilus syi were contminted (18). Cese nd Juzwik (5) recovered C. fgcerum from Colopterus trunctus less frequently thn from five other species (including Crpophilus syi) tht ccounted for 94% of ll beetles on ok wilt mts in My. Thus, not ll Colopterus trunctus nd Crpophilus syi exiting ok wilt mts re necessrily contminted. Proportions of C. fgcerum contminted beetles in wounds on helthy oks in stnd with ctive ok wilt in My lso were high (84% for Colopterus trunctus nd 71% for Crpophilus syi) (21). The proportions of dispersing sp beetles contminted with C. fgcerum in this study were much higher thn reported previously. Dispersing beetles trpped nd ssyed in Texs in the mid-1980s (1,2) nd erlier in Minnesot in the lte 1970s nd 1980s (18,19) were collected from fruit or fungus culture, nd/or fermenting flour dough bited trps. For exmple, only 0.65% of the 4,126 sp beetles in Texs (trpped October to My) nd 0.67% of the 1,043 sp beetles in Minnesot (trpped Mrch to June) yielded C. fgcerum. This difference my be ttributble to the use of ggregtion pheromones specific to the beetle species nd closely relted species. The temporl flight dynmics of dispersing Colopterus trunctus nd Crpophilus syi during the spring in stnds with ctive ok wilt nd the proportions found to be contminted with C. fgcerum further support the current thought tht these two species re the principl sp beetle vectors of the pthogen in Minnesot. Differences in flight dynmics lso suggest tht Colopterus trunctus my be reltively more importnt thn Crpophilus syi in this role. Furthermore, higher proportions of contminted beetles nd higher CIP vlues were found for Colopterus trunctus thn for Crpophilus syi in this study. These results, in turn, suggest Fig. 4. Contminted insect popultion estimtes for mle nd femle Colopterus trunctus (determined by multiplying the monthly fit men per site the proportion of individul insects tht tested positive for Certocystis fgcerum) for four sites by yer nd site type (ok wilt, A, 2002 nd C, 2003, or nonok wilt, B, 2002 nd D, 2003). Insects were trpped in wind-oriented funnel trps using species-specific pheromones from April to October in est centrl Minnesot Plnt Disese / Vol

9 tht the infective insect potentil is greter for Colopterus trunctus thn for Crpophilus syi, prticulrly during spring months. Bsed on his studies of numerous insect species ssocited with ok wilt mts nd infectble wounds on oks in Iow, Norris (26) lso concluded Colopterus trunctus is likely the most importnt sp beetle vector in tht stte, lthough quntittive nlysis ws not used to rech his conclusion. As previously indicted, CIP proved useful in our study to compre reltive importnce of two implicted sp beetle vector species. It lso my prove useful in compring two different vector groups, such s ok brk beetles (Coleopter: Scolytide) to sp beetles. Additionl fctors, (e.g., environmentl vribles nd mount of inoculum present) could be dded to risk model nd tested for mking such comprisons. Current ok wilt mngement guidelines in Minnesot include rting the risk of ok wilt trnsmission potentil by month during the yer (14). The guidelines specify tht pruning, wounding, or felling of oks during the high-risk period (April to June) should be voided, while the summer months re considered to be low-risk period, nd lte fll nd winter re considered sfe for such ctions. Although dispersing Colopterus trunctus nd Crpophilus syi were contminted with C. fgcerum during summer nd fll months, the CIP vlues were reltively low. However, CIP vlues were higher during the spring months. Thus, reltive risk in terms of infective insect potentil grees with previously published guidelines. The ttrctiveness of fresh summer nd fll wounds to Colopterus trunctus nd Crpophilus syi is not cler. Future studies will ddress the reltive ttrctiveness of fresh summer nd fll wounds to sp beetles, nd to Colopterus trunctus nd Crpophilus syi in prticulr. If C. fgcerum contminted beetles re found in fresh wounds during these sesons, the risk designtion will need to be ressessed. ACKNOWLEDGMENTS We thnk Frn Nelson, Vness Vldespino, nd Wendy Frncesconi for lbortory nd field ssistnce; Kthy Wrd for sttisticl nlysis ssistnce; Jordn Eggers for grphics ssistnce; nd Mrl Spivk, Steve Ktovich, Rob Doudrick, nd two nonymous individuls for review of the mnuscript. This work is portion of thesis by the first uthor in prtil fulfillment of M.S. degree requirements t the University of Minnesot. LITERATURE CITED 1. Appel, D. N., Andersen, K., nd Lewis, R Occurrence of Nitidulid beetles (Coleopter: Nitidulide) in Texs ok wilt centers. J. Econ. Entomol. 79: Appel, D. N., Kurdyl, T., nd Lewis, R Nitidulids s vectors of the ok wilt fungus nd other Certocystis spp. in Texs. Eur. J. For. Pthol. 20: Brnett, H. L Isoltion nd identifiction of the ok wilt fungus. W. V. Univ. Agric. Exp. Stn. Bull. 359T. Fig. 5. Contminted insect popultion estimtes for mle nd femle Crpophilus syi (determined by multiplying the monthly fit men per site the proportion of individul insects tht tested positive for Certocystis fgcerum) for four sites by yer nd site type (ok wilt, A, 2002 nd C, 2003, or non-ok wilt, B, 2002 nd D, 2003). Insects were cught in wind-oriented funnel trps using species-specific pheromones from April to October in est centrl Minnesot. Plnt Disese / October

10 4. Brtelt, R. J., Kyhl, J. F., Ambourn, A. K., Juzwik, J., nd Seybold, S. J Mleproduced ggregtion pheromone of Crpophilus syi, Nitidulid vector of ok wilt disese, nd pheromonl comprison with C. lugubris. Agric. For. Entomol. 6: Cese, K. R., nd Juzwik, J Predominnt nitidulid species (Coleopter: Nitdulide) ssocited with spring ok wilt mts in Minnesot. Cn. J. For. Res. 31: Cook, R. D., nd Weisberg, S Applied Regression Including Computing nd Grphics. John Wiley & Sons, New York. 7. Cosse, A. A., nd Brtlet, R. J Mle produced ggregtion pheromone of Colopterus trunctus: Structure, electrophysiologicl nd behviorl ctivity. J. Chem. Ecol. 26: Dorsey, C. K., Jewell, F. F., Lech, J. G., nd True, R. P Experimentl trnsmission of ok wilt by four species of Nitidulide. Plnt Dis. Rep. 37: Dorsey, C. K., nd Lech, J. G The bionomics of certin insects ssocited with ok wilt with prticulr reference to the Nitidulide. J. Econ. Entomol. 49: Dowd, P. F., Brtelt, R. J., nd Wicklow, D. T Novel insect trp useful in cpturing sp beetles (Coleopter: Nitidulide) nd other flying insects. J. Econ. Entomol. 85: Dowd, P. F., nd Nelsen, T Sesonl vrition of sp beetle (Coleopter: Nitidulide) popultions in centrl Illinois cornfield-ok woodlnd hbitt nd potentil influence of wether ptterns. Popul. Ecol. 23: Esker, P. D., nd Nutter, F. W., Jr Temporl dynmics of corn fle beetle popultions infested with Pntoe stewrtii, cusl gent of Stewrt s Disese of corn. Phytopthology 93: Freese, F Elementry Forest Smpling. U.S. Dep. Agric. Hndb French, D. W., nd Juzwik, J Ok Wilt in Minnesot. Univ. Minn. Ext. Serv. Publ. MI GO. 15. Gibbs, J. N., nd French, D. W The trnsmission of ok wilt. U.S. Dep. Agric. For. Serv. Res. Pp. NC Himelick, E. B., nd Curl, E. A Trnsmission of Certocystis fgcerum by insects nd mites. Plnt Dis. Rep. 42: Juzwik, J Fctors ffecting overlnd trnsmission of Certocystis fgcerum in Minnesot. Ph.D. thesis. University of Minnesot, St. Pul. 18. Juzwik, J., nd French, D. W Certocystis fgcerum nd C. picee on the surfces on free-flying nd fungus-mt-inhbiting nitidulids. Phytopthology 73: Juzwik, J., nd French, D. W Reltionship between Nitidulids nd Certocystis fgcerum during lte summer nd utumn in Minnesot. Plnt Dis. 70: Juzwik, J., Sklbeck, T. C., nd Neumn, M. F Nitidulids ssocited with fresh wounds on red oks during spring in Minnesot. (Abstr.) Phytopthology 89:S Juzwik, J., Sklbeck, T. C., nd. Neumn, M. F Sp beetle species (Coleopter: Nitidulide) visiting fresh wounds on helthy oks during spring in Minnesot. For. Sci. 50: Kyhl, J. F., Brtelt, R. J., Cosse, A. A., Juzwik, J., nd Seybold, S. J Semiochemiclmedited flight response of sp beetle vectors of ok wilt, Certocystis fgcerum. J. Chem. Ecol. 28: McMullen, L. H., Drke, C. R., Shenefelt, R. D., nd Kuntz, J. E Long distnce trnsmission of ok wilt in Wisconsin. Plnt Dis. Rep. 39: McMullen, L. H., Shenefelt, R. D., nd Kuntz, J. E A study of insect trnsmission of ok wilt in Wisconsin. Trns. Wisc. Acd. Sci., Arts Lett. 49: Merrill, W., nd French, D. W Insects nd the epidemiology of ok wilt. Pges in: Ok Wilt Perspectives. D. N. Appel nd R. F. Billings, eds. Proc. Ntl. Ok Wilt Sympos., Texs Agriculture Experiment Sttion, Texs Forest Service, nd Texs Agriculture Extension Service, Austin. 26. Norris, D. M Assocition of insects with the ok tree nd Endoconidiophor fgcerum Bretz. Ph.D. thesis. Iow Stte University, Ames. 27. Sklbeck, T. C The distribution of Nitidulide in deciduous forests of Minnesot. Ph.D. thesis. University of Minnesot, St. Pul Plnt Disese / Vol