Bur oak blight, a new disease on Quercus macrocarpacaused by Tubakia iowensis sp. nov.

Thomas C. Harrington1

Doug McNewHye Young Yun2

Department of Plant Pathology, 351 Bessey Hall,Iowa State University, Ames, Iowa 50011

Abstract: A newly recognized, late-season leaf diseaseof Quercus macrocarpa (bur oak) has become increas-ingly severe across Iowa and in neighboring statessince the 1990s. Vein necrosis and leaf death mayoccur over the whole crown or only on the lowerbranches. Symptoms typically intensify year-to-year inindividual trees, and there appears to be substantialvariation in susceptibility. Distinctive conidiomata(pycnothyria with a shield of radiating, setae-likehyphae) of a Tubakia sp. are found along the necroticleaf veins. The same species produces a second type ofpycnothyrium with a crustose covering and smallerconidia on the petioles of killed leaves, which remainon the tree through the winter and provide theprimary inoculum to infect newly emerging shootsand leaves in spring. Comparison of the Tubakia sp.on bur oak with T. dryina and other species ofTubakia led to the conclusion that the species on buroak is new, distinct from T. dryina, which herein isdefined more narrowly. Inoculation studies con-firmed that Tubakia iowensis sp. nov. is the cause ofbur oak blight. Bur oak blight appears to beparticularly severe on Q. macrocarpa var. oliviformis,which is well adapted to the dry, upland sites wherethe disease is found most frequently. The recentclimatic trend in Iowa to higher spring precipitationmight have led to increased severity of the disease.

Key words: climate change, Diaporthales, pyc-nothyria, Tubakia dryina

INTRODUCTION

A serious leaf disease of Quercus macrocarpa Michx.(bur oak or mossy-overcup oak) has been observed inIowa, southern Minnesota and eastern Nebraska sincethe mid-1990s (Anonymous 2002, Engelbrecht andFlickinger 2007). Symptoms of the disease, hereinnamed bur oak blight (BOB), include necrosis of the

leaf tissue along the veins and death of entire leafstarting in late July. Branches in the lower crown aregenerally the most severely affected, and severity ofthe disease tends to increase year-to-year in individualtrees. The most seriously affected trees typically showbranch dieback, perhaps associated with secondarywood borers, and some trees die after many years ofsevere BOB symptoms. The distinctive conidiomata ofa fungus tentatively identified as Tubakia dryina(Sacc.) B. Sutton (syn. Actinopelte dryina [Sacc.]Hohn.) are abundant along the necrotic veins.However the cause of the disease has not beenestablished.

In addition to being associated with leaf spots onmany species of oak (Quercus spp.), chestnut (Casta-nea spp.) and other tree genera in Europe, Japan andeastern North America (Glawe and Crane 1987,Munkvold and Neely 1990, Tehon 1948, Yokoyamaand Tubaki 1971), species of Tubakia are known asleaf and twig endophytes in oaks (Cohen 1999,Gonthier et al. 2006, Hashizume et al. 2008, Sieber2007), and they commonly sporulate on tissues killedby other agents (Munkvold and Neely 1990). Resultsof pathogenicity tests with species of Tubakia oftenhave been ambiguous (Holdenreider and Kowalski1989, Yokoyama and Tubaki 1971), although Munk-vold and Neely (1990), Zhang and Walker (1995),Taylor and Clark (1996) and Kowalski (2006) weresuccessful in producing leaf spot symptoms oninoculated oaks and sweetgum (Liquidambar styraci-flua L.). T. dryina has been associated with leaf spotson many species of oak in eastern North America(Glawe and Crane 1987, Limber and Cash 1945,Munkvold and Neely 1990), but until recently bur oakwas known as a host only through four herbariumspecimens from Wisconsin, Illinois and Virginia(Glawe and Crane 1987).

In addition to T. dryina four Japanese species ofTubakia are recognized (Sutton 1973, Yokoyama andTubaki 1971). However mycologists and pathologistsin Europe and eastern USA have taken a broad viewof T. dryina and all species that form the character-istic conidiomata of Tubakia have been consideredsynonyms of T. dryina (Glawe and Crane 1987,Limber and Cash 1945, Tehon 1948). The conidio-mata of Tubakia spp., generally referred to aspycnothyria, consist of a columella-like structurecovered by a convex shield (scutellum) of a radiatingseries of pigmented, thick-walled cells (Munkvold and

Submitted 15 Jul 2011; accepted for publication 18 Jul 2011.1 Corresponding author. E-mail: tcharrin@iastate.edu2 Present address: Systematic Mycology and Microbiology Laborato-ry, USDA-ARS, Beltsville, Maryland 20705

Mycologia, 104(1), 2012, pp. 79–92. DOI: 10.3852/11-112# 2012 by The Mycological Society of America, Lawrence, KS 66044-8897

79

Neely 1991, Taylor 2001). Production of new conidiafrom the underside of the developing scutellumpushes the older conidia out from under theprotection of the scutellum, presumably allowingrain-splash dispersal. A teleomorph for T. dryina,Dicarpella dryina Belisario & ME Barr (Diaporthales)was described from seedlings of red oak (Quercusrubra L.) in a nursery in Italy (Belisario 1991), but noother teleomorph connection has been made forspecies of Tubakia.

Investigations of the new bur oak disease began in2008 when it became apparent that there are a numberof Tubakia species on bur oak and other oak species inIowa. A fungus that matched the description of T.dryina was found on white oak (Q. alba L.) and rarelyon bur oak, but the Tubakia species associated with buroak blight appeared to be distinct. This paper gives adescription of BOB, completes Koch’s postulates toshow that an undescribed Tubakia species is the cause,describes the pathogen as a new species and comparesand contrasts the new species with T. dryina.

MATERIALS AND METHODS

Disease observations, specimen and isolate collection.—Treeswith symptoms of BOB were observed in natural standsacross Iowa and into neighboring states, but most diseaseobservations were made 2008–2010 at two sites with grovesof mature (. 100 y old) bur oak in Ames, Iowa. BrooksidePark is bottomland near Squaw Creek, where only a few buroak trees were severely affected by BOB. The uplandWestwood site had a somewhat open, former-savanna grovewith many severely affected bur oak trees.

Symptomatic and asymptomatic twigs and leaves werecollected for microscopic examination and isolation at-tempts. Green, asymptomatic leaves from diseased andhealthy trees were placed in large Petri dishes withmoistened paper towels, and the plates were positionednear a south-facing window for development of lesions and/or conidiomata. Separate plant tissue was surfaced-sterilizedwith 100% bleach for 3 min with stirring, followed by 30 s in95% ethanol and rinsed in sterile water before plating onmalt extract agar (MEA, 1.5% malt extract and 2% agar).

Conidiomata of Tubakia spp. were dislodged easily fromleaf blades or veins by scraping with a dissecting needle.Isolations were made by streaking several conidiomata withconidia in a drop of water across the surface of agar (MYEA,2.0% malt extract, 0.2% yeast extract and 2.0% agar)amended with 100 mg/L streptomycin sulfate. Subculturesof developing colonies were made after 1–3 d.

Dead leaves on and under trees with BOB were collectedin winter and spring and incubated under humid condi-tions in the laboratory for production of perithecia andconidiomata. Isolations were attempted by crushing peri-thecia and streaking out ascospores on MYEA. Leaves fromthe previous season still on branches of diseased bur oaktrees also were examined, and conidia from conidiomata onpetioles were streaked onto MYEA plates for isolation.

Symptomatic leaves of bur oak and other Quercus spp.were examined from across Iowa and neighboring states.Many of the samples were submitted by homeowners andforesters with the Iowa Department of Natural Resources(DNR) to the Iowa State University (ISU) Plant and InsectDiagnostic Clinic. Plant diagnosticians from neighboringstates also submitted material for study. Specimens from theUS National Fungus Collection (BPI), the Illinois NaturalHistory Survey (ILLS) and the Ada Hayden Herbarium(ISC) at ISU were examined. Cultures of Tubakia spp. wereobtained from the Centraalbureau voor Schimmelcultures(CBS, Utrecht, the Netherlands). Culture numbers begin-ning with A are those of the senior author, and represen-tative cultures were deposited in CBS (TABLE I). Type andrepresentative specimens were deposited in ISC and BPI.

Characterization of specimens and isolates.—Isolates weregrown on MYEA plates at room temperature (21–23 C) or atset temperatures (16, 20, 25, 30 and 35 C). To comparegrowth rates a plug of mycelium 4 mm diam from theadvancing margin of a 7 d old colony was placed on a MYEAplate, and the radial growth (mm/d) was determined 2–7 d.Three replicate plates were used for each isolate/temper-ature combination. For formal description cultures weregrown on MYEA at 25 C in a dark incubator.

Fungal material from plants and cultures was mounted in20% lactic acid or in lactophenol with cotton blue formicroscopic examination. Digital photographs and mea-surements and were taken on an Olympus compoundmicroscope (BH-2, Nomarski DIC optics) fitted with adigital camera (DFC295) and software from Leica (Ban-nockburn, Illinois).

DNA sequencing.—Sequencing of portions of rDNA wereattempted from DNA extracted from the mycelium fromthe surface of MYEA plates with PrepManTM Ultra (AppliedBiosystems, Foster City, California). The LSU (largesubunit, 26S) rDNA region was amplified with primersLROR and LR5, and the PCR products were sequenced withprimers LROR and LR3 (Harrington et al. 2010). The ITS-rDNA amplifications used primers ITS1-F and ITS-4, andthe PCR products were sequenced with the same primers(Harrington et al. 2002).

Comparisons to other rDNA sequences were conductedwith BLAST 2.2.24 queries (National Center for BiotechnologyInformation, National Institute of Health, Bethesda, Mary-land). Representative sequences were deposited in GenBank.

Inoculation experiments.—Three representative Tubakiaisolates from blighted bur oak trees at the Westwood sitewere selected for use in four inoculation experiments(TABLE I). Isolates of three other Tubakia spp. wereincluded in the third experiment (TABLE I). Isolates ofthe BOB pathogen do not sporulate abundantly on MYEA,so all isolates were cultured at room temperature onautoclaved bur oak leaves on wet filter paper in a largeplastic Petri dish placed near a south-facing window 2 wk.Conidia produced from sporodochia on the leaves weremixed with approximately 1 mL 0.02% Tween 80 (FisherScientific, Pittsburgh, Pennsylvania) and withdrawn with apipette tip. Conidia concentrations were determined with a

80 MYCOLOGIA

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HARRINGTON ET AL.: TUBAKIA IOWENSIS SP. NOV. 81

FIG. 1. Signs and symptoms of bur oak blight (BOB) on bur oak. A. Almost all leaves on two trees on the left have died, butother bur oak show few or no symptoms (early Sep 2008). B. Tree in center recently dead and bur oak trees on either side havelost most of their leaves due to defoliation (17 Sep 2010). C. Branch dieback on a tree suffering several years of BOB. D.Purple-brown discoloration on midvein and major lateral veins on the underside of a leaf (14 Jun 2010). E and F. Necrosis of

82 MYCOLOGIA

haemacytometer and adjusted to 3.9–4.2 3 106 per mL inthe first experiment, to 3.8 3 106 per mL for isolates A573and A578 and 1.4 3 106 per mL for isolate A668 in thesecond experiment, from 3.5–4.8 3 106 for the thirdexperiment and 2.3–2.6 3 106 conidia/mL in the fourthexperiment. Autoclaved bur oak leaves were washed with0.02% Tween 80 for control inoculum.

For test plants 2 y old, bare-root bur oak seedlings werepurchased from the Iowa DNR nursery in Ames in late 2008and stored refrigerated until planting in 10 3 10 3 35 cmplastic pots with a mixture of peat moss, coarse perlite andpremixed soil (Sunshine SB300 UNIVERSAL, Sun GroHorticulture, Vancouver, Canada) in a 2 : 4 : 1 ratio.

In the first three experiments seedlings were planted 16,17 Jul 2009; inoculations followed respectively at 45 d, 66 dand 116 d post-planting. Each of three seedlings had threewounded leaves (three pseudoreplicates) that were inocu-lated with the same isolate. In the second and thirdexperiments each of the seedlings also had three unwound-ed leaves inoculated with the same isolate. For woundinoculations the midvein of the abaxial side was wounded at6–8 spots with a sterile needle (Precisionglide, 27G K, BD,Franklin Lakes, New Jersey) at intervals of 1.5 cm, startingat 1.5 cm above the petiole. Wounded or unwoundedmidveins were brushed with the spore suspension of one ofthe isolates or with control inoculum. Seedlings inoculatedwith the same isolate were placed in a crate afterinoculation, sprayed with a fine mist and covered with awhite, polyethylene bag 48 h. The seedlings were uncoveredand incubated in a greenhouse at 25–28 C with supplemen-tal lighting. Seedlings were inspected initially for symptomseach 1–3 d, but some inspection intervals were as long as 7 dtoward the end of the experiments. All inoculated leavesthat died were collected, and all wound-inoculated orcontrol leaves remaining on the seedlings were collectedafter 54 d of the first experiment, after 76 d in the secondexperiment and after 63 d in the third experiment.Unwounded leaves were inspected respectively up to 99 dand 75 d in the second and third experiments. Isolationswere attempted from at least one killed leaf from eachseedling as well a leaf from each control seedling. Leafsections were surface-sterilized 1 min in 95% ethanol, rinsed1 min in distilled water and plated on MYEA.

In the fourth inoculation experiment dormant bare-rootseedlings were planted Feb 2010 and inoculated 21 d lateron 3 Mar. The young, green twig tissue and/or petioles of13 seedlings were inoculated (five with A573, five with A578,and three control seedlings) by brushing on the conidial

suspension. The seedlings were misted with water andcovered as described above for 48 h. Seedlings wereinspected at 1–7 d intervals for symptom development forup to 56 d. On 23 Sep the seedlings were moved outside toallow for normal senescence of leaves, which was completeduring the first week of Nov.

RESULTS

Field symptoms and distribution.—Conspicuous leafsymptoms with many dead leaves hanging on lowerbranches or throughout the crown were apparentafter mid-Jul 2008 and 2009 in Ames, but some BOBsymptoms were seen in early Jul 2010. By late Aug2008 and 2009 most leaves on some trees were deadand hanging on branches, but even in the mostseriously affected groves less than one-third of thetrees showed severe symptoms (FIG. 1A). In 2010, ayear with heavy mid- to late-summer rainfall (NOAA2011), many infected leaves were shed in mid-Julythrough August, and many of these trees had sparsefoliage due to defoliation (FIG. 1B). Disease severitytended to increase in individual trees year-to-year.Branch dieback typical of that associated with the two-lined chestnut borer, Agrilus bilineatus (Weber)(Haack and Acciavatti 1992), was seen in many ofthe most severely affected trees (FIG. 1C). At theWestwood site a few trees with severe BOB symptomsthe previous fall failed to produce leaves and died inspring 2009, 2010 and 2011. Most of those trees hadsubstantial branch dieback in previous years. Asidefrom branch dieback, most surviving trees with BOBproduced normal, healthy-appearing leaves in thespring of the next year.

Initial leaf symptoms consisted of small, purple tobrown spots on the veins on the underside side ofleaves in June (FIG. 1D). Small necrotic spots on theblade tissue also were evident on some leaves. By mid-July lesions expanded along the veins and coalescedand vein necrosis was evident on the upper and lowersurfaces of the leaves (FIG. 1E, F). When two necroticveins met the intervein tissues or the distal portion ofthe leaf blade became chlorotic and died. Conidio-mata of a Tubakia sp. were abundant on and along

r

midvein and major lateral veins (mid- and late-Jul 2009, respectively). G. Conidiomata along the midvein and a lateral vein ontop side of leaf. H. Green leaf with necrotic (brown) petiole and black, crustose, immature conidomata (6 Jul 2010). I.Recently killed leaves still hanging on branches and newly-flushed green leaves (27 Aug 2007, photo by A. Flickinger). J. Dead,overwintering leaves (25 Feb 2009). K. Black, subepidermal conidiomata on the petiole of an overwintering leaf. Arrowindicates site where leaf abscission should have taken place. L. Wound-inoculated leaf showing necrosis surrounded bychlorosis along an inoculated vein (black arrows) 30 d after inoculation. M. Black spots and pustules on petiole and midveinon the underside of a bur oak leaf inoculated 104 d earlier by placing a conidial suspension on the undifferentiated petiole. N.Cultures of Tubakia iowensis (left) and T. dryina (right) on malt yeast extract agar.

HARRINGTON ET AL.: TUBAKIA IOWENSIS SP. NOV. 83

the necrotic veins on the upper leaf surface (FIG. 1G)and on the veins on the lower leaf surface. As thenecrosis progressed down the midvein to the petiole,the leaf often died and fell off the tree. Suchdefoliation was particularly noticeable in late 2010(FIG. 1B).

Some leaves developed black pustules at the base ofthe petioles in mid- to late August (FIG. 1H). Thesepetioles were green or had necrosis near the point ofattachment to the twig. Leaves with black pustuleseventually died but typically remained attached to thetwigs (FIG. 1I, J). Nearly all leaves died on some trees(FIG. 1A), and many of these trees flushed new leavesin August (FIG. 1I). After normal leaf fall, when healthybur oak trees had lost all of their leaves, many of thekilled leaves on BOB trees remained on the twigs andpersisted into spring (FIG. 1J). These leaves were firmlyattached, although an abscission layer was visible, andblack pustules developed under the epidermis on thelower petiole and on the twig immediately below theabscission layer (FIG. 1K). Next spring, especiallyduring rainy periods, the black pustules swelled andconidia developed underneath the pustules.

Most of the trees with severe BOB symptoms were. 100 y old and 50 cm diam (1.4 m tall) in remnantsof savanna forests in small groves or in relatively openstands. The disease appeared to be much more severeon upland than on bottomland sites. Mature bur oaktrees in dense, mixed-hardwood stands showed few orno symptoms of BOB. The disease was particularlysevere in eastern Nebraska and western and centralIowa. We were unable to find the disease in the farsoutheastern corner of Iowa. A few isolated bur oaktrees with BOB symptoms were observed across theborder in western Illinois and northern Missouri. Buroak leaves with BOB symptoms were sent to us fromnortheastern Kansas, southern Minnesota and south-western Wisconsin in 2010.

Isolations.—Conidia from conidiomata on or along-side veins of diseased bur oak leaves and onoverwintering petioles yielded a fungus with moder-ate growth rate, white to light gray mycelia, scallopedmargins and concentric rings of dense aerial myceli-um (FIG. 1N). Similar isolates were obtained fromasymptomatic leaves, petioles and fresh shoots col-lected in May and June from trees that had BOBsymptoms the previous summer. The fungus also wasisolated from a green acorn taken from a tree withBOB. Occasionally similar isolates were obtained fromsurface-sterilized, 1 or 2 y old, asymptomatic twigs,although another species (Tubakia sp. A) was morecommonly isolated from living twigs of bur oak.

Isolations were attempted from other Quercus spp.with leaf spots and necrotic leaf veins, but with only

one exception (from an asymptomatic petiole of Q.rubra) the new Tubakia sp. only was isolated from buroak. Specimens and cultures of the new Tubakia sp.were identified from 57 of Iowa’s 99 counties andfrom five of six neighboring states and Kansas. Buroak trees in South Dakota were not examined.

The LSU-rDNA sequence of the new Tubakia sp.was closest to that of an isolate from a dead leaf of Q.rugosa Nee in Arizona (HM122939, 538 of 539 bpmatching), Apiognomonia supraseptata S. Kaneko &Ts. Kobay. (AF277127, 537 bp matching) and afungus from a sweetgum seedling in North Carolina(GU552498, 533 bp matching). The ITS-rDNAsequence of the new species was closest to those of aDicarpella sp. from Q. rubra in New York (HM855225and HM855226, 504 of 524 bp matching), unidenti-fied fungal endophytes (AY546028 and GQ996086,502 and 500 bp matching respectively) and isolatesdeposited in CBS as D. dryina (AY853241, AY853240and FJ598616, with 501, 500 and 494 bp matchingrespectively). There was only minor variation in theITS-rDNA sequences (one site in the middle of ITS 1and another near the end of ITS 2) among 95 isolatesand specimens of the new Tubakia sp.

Other Tubakia spp., including one matching thedescription of T. dryina, were isolated from leaves ofbur oak and other Quercus spp. with vein necrosis ordiscreet spots or from asymptomatic twigs (endo-phytes). Although difficult to separate based onpycnothyria and conidia on leaves, the other Tubakiaspp., especially T. dryina (FIG. 1N), were readilydistinguished from the BOB Tubakia by rDNAsequences (TABLE I), growth rates, pigmentationand capacity to sporulate on MYEA. Tubakia sp. Awas isolated commonly from healthy twigs, leaves andacorns of bur oak and had an ITS-rDNA sequencesimilar to that of T. dryina. Tubakia sp. B was isolatedfrom bur oak and other members of the white oakgroup (Quercus L. sect. Quercus) and had ITS-rDNAsequences that differed from the BOB pathogen atone base position. Another species tentatively identi-fied as Actinopelte americana Hohn. (Hoehnel 1925)was isolated occasionally from leaf spots and veinnecrosis of bur oak, but it was isolated much morefrequently from Q. rubra and other species from thered oak group (Quercus sect. Lobatae Loudon).

Perithecia commonly formed along the veins ofincubated leaves collected in winter and early springfrom under bur oak trees that showed BOB symptomsthe previous summer, but isolations from ascosporesyielded only an Apiognomonia sp. or other relatedfungi, which may be endophytes and/or cause oakanthracnose (Sinclair and Lyon 2005). No sexual stateof a Tubakia sp. was encountered. Conidiomata ofTubakia spp. were not seen on leaves collected during

84 MYCOLOGIA

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HARRINGTON ET AL.: TUBAKIA IOWENSIS SP. NOV. 85

the winter or early spring from the ground undertrees that had BOB symptoms.

Inoculation experiments.—In the first inoculationexperiment all but one leaf inoculated with A573and the control leaves developed vein necrosis(TABLE II). Chlorotic spots on the midvein underthe leaf were seen at the point of inoculation within2 d and turned purplish at 2–5 d. Reddish brownnecrosis surrounded by chlorosis was visible on theupper leaf surface by day 9 (FIG. 1L). As the necrosisexpanded down the leaf to the petiole the leaf usuallydied and fell off the seedling (TABLE II). The BOBTubakia sp. was isolated from all inoculated leaves.None of the control leaves developed symptoms ordied, and the Tubakia sp. was not isolated from them.

In the second inoculation experiment progress ofsymptoms on wound-inoculated leaves was similar tothat of the first experiment (TABLE II). Purple lesions,vein necrosis on the top side of the leaves and leafdeath developed more slowly on the unwoundedleaves than on the wounded leaves (TABLE II). Allunwounded leaves inoculated with A573 showedsymptoms and eight leaves died (at an average of66 d post inoculation). For A578 the eight leaves thatdied did so after an average of 80 d. All leavesinoculated with A668 showed symptoms, but only twoleaves on one of the three inoculated seedlings died.None of the control inoculations showed symptoms.The inoculated fungus was recovered from inoculatedleaves but not from control leaves.

The third experiment used older leaves, and theseedlings inoculated with the BOB isolates developedsymptoms more quickly, with death of unwoundedleaves occurring 20 d sooner than in the secondexperiment (TABLE II). Isolates of three other Tuba-kia spp. caused the same symptoms as the BOBisolates. The inoculated Tubakia spp. were recoveredin all isolation attempts.

In the fourth experiment inoculation of developinggreen shoots and petioles with A573 and A578 did notresult in vein necrosis characteristic of BOB. Howeverthe 18 green shoots on five seedlings that wereinoculated with A573 showed necrosis at 5–30 d, 10 ofthose shoots developed small black pustules at 13–40 d, 13 petioles developed black pustules at 5–40 dand eight leaves developed black pustules on themidveins at 6–40 d (FIG. 1M). Of the three petiolesinoculated with A573, one leaf had black spots on themidvein by day 13, one leaf developed black spots onthe petiole at 13 d and on the midvein at 30 d, andone of the developing leaves died at 20 d. Of the 12green shoots on five seedlings inoculated with A578,10 developed black spots on the shoots at 5–10 d, fiveof these developed small black pustules at 13–30 d, six

developed black spots on the petiole at 5–13 d andtwo developed black spots on the midvein on theunderside of the leaves at 16 d. Of the five petiolesinoculated with A578, all developed black spots at 3–13 d, two of these developed black pustules on thepetiole and four developed black spots on themidvein at 13–16 d.

The seedlings from the fourth experiment wereplaced outside in September and shed their leaves bythe first week of November. However three dead leaves(from young shoots inoculated with A573) on one ofthe seedlings remained attached and small blackpustules developed on the petioles of these leaves.The blade of one of the three leaves was collected,surface-sterilized and plated onto MYEA with strepto-mycin, but no Tubakia sp. was isolated. However thepetiole of this leaf was wetted and incubated undermoist conditions and microscopic examinationshowed conidia production under the black pustule.Isolations from those conidia and from the surface-sterilized petiole yielded the new Tubakia sp.

TAXONOMY

Tubakia iowensis T.C. Harr. & D. McNew sp. nov.FIG. 2

MycoBank MB561043Pycnothyrii en lamina epiphylli et hypophylli, brunnei

cum scutelli asterinei, 40–110(135) mm diam, conidii hyaliniad brunnei, eseptati, obovati ad subglobosi, 9–14.5 3 7.5–10.5 mm. Pycnothyrii en petiolum nigelli, 150–500 mm diam,cum scutelli crustacei, conidii hyalini ad brunnei, eseptati,obovati ad ellipsoidei, 9.5–14 3 6.5–8.5 mm.

Conidiomata (pycnothyria) with radiate scutellaepiphyllous or hypophyllous on Quercus macrocarpa,forming primarily on or along the midvein and majorlateral veins (FIG. 2A). Scutella convex, of brown, thick-walled cells radiating from a central point, rounded topointed at the tips (FIG. 2B), 40–110(–135) mm diam.Conidiophores short, forming under the developingscutellum (FIG. 2C, D). Conidia thick-walled, smoothto finely verrucose (FIG. 2D), initially hyaline, laterbrown, obovoid to subglobose, 9.0–14.5 3 7.5–10.5 mm.Microconidia sometimes produced from the samepycnothyrium as macroconidia or from smaller pyc-nothyria, hyaline, fusiform, curved, 4–8.5 3 1–2 mm(FIG. 2E). Sporodochia a cluster of conidiophores,hypophyllous, forming on the midvein or major lateralveins (FIG. 2F), irregular, sometimes with small, poorlydeveloped scutella. Conidia of sporodochia lightbrown to dark brown en masse, same size and shapeas conidia from pycnothyria with radiate scutella.

Crustose conidiomata (pycnothyria) on overwinter-ing leaves at base of petioles, or just below on twigs,subepidermal, black, irregular, 150–500 mm diam,

86 MYCOLOGIA

FIG. 2. Conidiomata and conidia of Tubakia iowensis on bur oak leaves. A. Black pycnothyria with radiate scutella on andalongside the midvein on the upper leaf surface. B. Top view of radiate scutellum. C and D. Bottom view of radiate scutellumshowing conidia produced from short conidiophores radiating from the center (conidiophores lower left in D). E.Microconidia from pycnothyrium (9 Sep 2009). F. Dark conidial masses from sporodochia on a midvein on the undersurfaceof a leaf. G. Crustose pycnothyria developing on a petiole (30 Dec 2010). H. Erumpent, mature pycnothyria on petiole (1 May2009). I. Longitudinal section of a developing, crustose pycnothyrium (from 2G) with conidiophores on the underside of thescutellum (upper left) and on subepidermis (bottom right). J. Longitudinal section of a mature, crustose pycnothyrium (from2H) with black scutellum (s) bursting through the epidermis (e) due to development of conidia (c). K. Conidia from acrustose pycnothyrium (from H). L. Subepidermal scars from dehiscence of crustose pycnothyria on petiole (21 Jun 2009).FIGS. 2A to D and F from holotype (12 Aug 2008); 2E from ISC 448603; 2H, J and K from ISC 448601, which, along with 2G, I,and L, are from petioles on the same tree as the holotype.

HARRINGTON ET AL.: TUBAKIA IOWENSIS SP. NOV. 87

sometimes confluent (FIG. 2G, H). Conidiophoresshort, forming in the winter or spring on theunderside of the developing scutellum and on theplant surface under the scutellum (FIG. 2I, J). Conidiathick-walled, finely verrucose to smooth, initiallyhyaline, later brown, obovoid to ellipsoidal, 9.5–143 6.5–8.5 mm (FIG. 2K). Scutella of thick-walled cells,textura angularis, dehiscing marginally or by irregularfissures. Subepidermal scars from dehisced pyc-nothyria evident on petioles (FIG. 2L)

Colonies with optimal growth at 25 C on malt yeastextract agar, attaining 50–56 mm diam after 7 d, nogrowth or trace at 35 C., with a scalloped margin(FIG. 1N), white at first, felt-like and light gray at 10 d,with concentric rings of dense aerial mycelium,underside yellow at 7 d, lighter toward the edge ofthe colony, golden to yellow at 10 d, with dark gray,dense, tissue in concentric rings. Conidia in culturerare, thick-walled, smooth to finely verrucose, initiallyhyaline, later brown, variable size and shape, obovoidto ellipsoidal, 8.5–15.5(18.5) 3 5–8(8.5) mm.

Specimens examined. USA. IOWA: Ames, Brookside Park,leaves of Q. macrocarpa, 21 Aug 2008, T. Harrington, ISC448599 (HOLOTYPE), BPI 881219 (ISOTYPE); petioles ofQ. macrocarpa, 1 May 2009, D. McNew, ISC 448601; 16 Nov

2010, D. McNew, ISC 448602; leaves of Q. macrocarpa, 9 Sep2009, D. McNew, ISC 448605; 30 Sep 2009, D. McNew, ISC448607; 9 Sep 2009, D. McNew, ISC 448603; petioles of Q.macrocarpa, 21 May 2008, S. Kim, ISC 448604; DecaturCounty, petioles of Q. macrocarpa, 18 Mar 2010, D. McNew,ISC 448606; Decorah, leaves of Q. macrocarpa, 24 Sep 2009,D. McNew, ISC 448609; Monona County, leaves of Q.macrocarpa, 21 Aug 2008, A. Flickinger, ISC 448614; JasperCounty, leaves of Q. macrocarpa, 1 Oct 2009, L. Mottl, ISC448608. WISCONSIN: Monroe, leaves of Q. macrocarpa,Oct 2010, K. Scanlon, ISC448610.

Cultures examined. USA. IOWA: Ames, Brookside Park,leaf of Q. macrocarpa, 21 Aug 2008, T. Harrington, CBS129012 (A607) (ex HOLOTYPE); Ames, from ISC 448602,CBS 129019 (A1003). WISCONSIN: Monroe, from ISC448610, CBS 129017 (A995).

Tubakia dryina (Sacc.) B. Sutton, Trans. Brit. Mycol.Soc. 60:165. 1973 FIG. 3; Leptothyrium dryinum Sacc., Michelia 1:202. 1878.; Actinopelte dryina (Sacc.) Hohn., Mitteilunguen aus

dem botanischen Institut der Technischen Hochschulein Wien. 2:69. 1925.

Conidiomata (pycnothyria) on leaves of Quercusspp. Scutella convex, of brown, thick-walled cellsradiating from a central point, rounded to pointed at

FIG. 3. Conidiomata and conidia of Tubakia dryina sensu stricto. A and B. Pycnothyria with radiate scutella. C and D.Conidia from pycnothyria with radiate scutella. E. Conidia from culture on malt yeast extract agar. F. Conidia from crustosepycnothyrium. G. Twig of Quercus alba with erumpent, crustose pycnothyrium (right arrow) and another pycnothyriumpushing through the twig epidermis (left arrow). Conidia in E and F stained with cotton blue in lactophenol. FIGS. A and C arefrom isotype specimen BPI 391924 collected in Treviso, Italy. Other figures are from Ames, Iowa: B and D from ISC 448612, Efrom isolate A789, and F and G from ISC 448611.

88 MYCOLOGIA

the tips (FIG. 3A, B), 60–110(125) mm diam. Conid-iophores short, forming under the developing scutel-lum. Conidia thick-walled, smooth to finely verrucose,initially hyaline, later brown, obovoid to ellipsoidal,10–15 3 7.0–10.5 mm (FIG. 3C, D). Microconidiasometimes produced from the same pycnothyrium asmacroconidia or from smaller pycnothyria, hyaline,fusiform, 4–9 3 1.5–2.5 mm. Crustose conidiomata(pycnothyria) on twigs erumpent (FIG. 3G), black,irregular, 0.2–1 mm diam. Crustose scutella black,dehiscing by irregular fissures. Conidiophores short,on the underside of the developing scutellum and onthe plant surface under the scutellum. Conidia fromcrustose pycnothyria thick-walled, smooth to finelyverrucose, initially hyaline, later brown, obovoid toellipsoidal, 10–16 3 6–7.5 mm (FIG. 3F). Colonies withoptimal growth at 25 C on malt yeast extract agar,attaining 60–66 mm diam after 7 d. Mycelium with ascalloped margin (FIG. 1N), creamy white at first, withconcentric rings of aerial mycelium, underside darkgray in the middle, medium brown to yellow towardthe edge at 10 d. Sporodochia abundant at 10 d, inconcentric rings, conidial masses dark gray to black.Conidia from sporodochia in culture thick-walled,finely verrucose, initially hyaline, later brown, obovoidto ellipsoidal, 10–16 3 5.5–8.5 mm (FIG. 3E).

Specimens examined. ITALY. VENETO: Treviso, BoscoMontello, leaf spots on Quercus pedunculata (5 Q. robur),Sep 1875, in Saccardo, Mycotheca Veneta No. 555, BPI391920 (ISOTYPE); Conegliano, leaf spots on Q. pseudoru-bra (5 Q. petraea), 1878, Spegazzini, in Thumen, MycothecaUniversalis Cent. 16, 1584, BPI 391924. USA. IOWA: Ames,Pammel Woods, twig of Q. alba, 23 Mar 2010, D. McNew,ISC 448611; Muscatine County, leaf spots on Q. macrocarpa,2 Aug 2010, D. McNew, ISC 448612; Sigourney, leaf spots onQ. alba, 23 Aug 2010, D. McNew, ISC 448613.

Cultures examined. ITALY. VENETO: Treviso, FagareForest, Q. robur, S. Mutto-Accordi, CBS 112097. NEWZEALAND: Auckland, Mount Albert, leaf spot on Q. robur,11 Nov 2003, R. Afford, CBS 114386. USA. IOWA: fromISC448611, CBS 129016 (A876); from ISC 448612, CBS129018 (A996); Ames, from leaf spot on Q. alba, 16 Aug2009, D. McNew A789.

Tubakia specimens from eastern North Americahave been considered T. dryina because of thesimilarity of the size of pycnothyria and conidia(Glawe and Crane 1987, Limber and Cash 1945,Tehon 1948), but examinations of cultures and DNAsequences from Quercus spp. in Iowa and elsewhereindicate that T. dryina is a complex of species. SomeIowa isolates from Q. macrocarpa, Q. alba and Q. roburfit the description of T. dryina from Europe, andthese isolates have identical ITS-rDNA and LSU-rDNAsequences (TABLE I). No sequence is available fromSaccardo’s isotype material (BPI 391920) on Q. robur(FIG. 3A, C) or from the specimen (BPI 391924)

considered to be authentic by Hoehnel (1925), butboth specimens morphologically match the emendeddescription for T. dryina given above. Both Italianspecimens were collected near Treviso, as was a laterisolate from Q. robur (CBS 112097), which has the T.dryina ITS-rDNA sequence, as does an isolate (A841)from a crustose pycnothyrium on a twig of Q. robur inGermany (Holdenreider and Kowalski 1989) and anisolate (CBS 114386) from Q. robur in New Zealand(TABLE I). Descriptions of pycnothyria (30–50 and70–135 mm diam respectively) and conidia (12–15 3

5–8 and 9.5–15.5 3 5–8.5 mm respectively) of T.dryina specimens from Japan (Yokoyama and Tobaki1971, Kobayashi et al. 1979 respectively) do not matchthe narrow concept of T. dryina.

The radiate scutella and associated conidia of T.iowensis are difficult to distinguish from those of T.dryina. However the crustose pycnothyria of T. dryinaare larger and their conidia narrower than thecrustose pycnothyria and conidia of T. iowensis.Conidia of both species produced from crustosepycnothyria are narrower than conidia from pyc-nothyria with radiate scutella, and conidia producedin culture are more variable in size and shape.Cultures of the two species differ markedly: T. dryinais much darker than T. iowensis and producesabundant sporodochia and conidia. Furthermoreisolates of T. dryina (A768, A890, A942) grew fasterat 25 C (4.5–4.8 mm/d) and at 30 C (3.9–4.6 mm/d)on MYEA than did isolates of T. iowensis (A578, A607,A874, A908), which grew 3.7–4.3 and 3.2–3.8 mm/dat those respective temperatures. The ITS-rDNAsequences of T. iowensis and T. dryina also differ(only 502 of 523 bp matching).

Based on our isolations T. iowensis occurs almostexclusively on Q. macrocarpa. In contrast T. dryinaoccurs on many species of the white oak group. Otherspecies of Tubakia, some undescribed (e.g. Tubakiasp. A and B) and one similar to A. americana(Hoehnel 1925), were found on bur oak and otherQuercus spp. in Iowa and surrounding states. The fourherbarium specimens from bur oak collected in the1950s from Wisconsin and Illinois and a 1945collection from Virginia (TABLE I) had discrete leafspots that were atypical for BOB. The size and shapeof the scutella and conidia on the specimens fromWisconsin and Illinois do not differ from those ofTubakia sp. B, but without cultures or rDNAsequences we are not able to definitively identifythese collections. The Virginia specimen also differsfrom T. iowensis; it might be A. americana.

The only teleomorph described for a Tubakiaspecies, Dicarpella dryina (Belasario 1991), was foundin a nursery in Italy on seedlings of red oak (Quercusrubra), a North American oak in sect. Lobatae, a

HARRINGTON ET AL.: TUBAKIA IOWENSIS SP. NOV. 89

section that does not naturally occur in Europe orAsia. A culture from ascospores of D. dryina (CBS639.93) appears to be A. americana or among a groupof closely related Tubakia spp. in North America thatare found on sect. Lobatae. Thus it appears that D.dryina is not the teleomorph of T. dryina or T.iowensis. Microconidia of T. iowensis and T. dryinaobtained from pycnothyria with radiate scutella didnot germinate on MYEA, and it is assumed that theyserve as spermatia, suggesting that these two specieshave a teleomorph.

DISCUSSION

The well known endophyte and leaf spot pathogen onoaks, T. dryina, initially was thought to be the cause ofthe newly recognized leaf blight on bur oak, but wehave found at least five species of Tubakia producingpycnothyria with radiate scutella in leaf spots or alongnecrotic veins of Q. macrocarpa in Iowa. Seedlinginoculations demonstrated that each of these Tubakiaspecies is capable of inducing vein necrosis in buroak, but only T. iowensis is associated with severe leafblight on bur oak, and its conidiomata are found onalmost every blighted leaf; also only T. iowensisproduces crustose pycnothyria on the petioles ofoverwintering leaves.

Crustose pycnothyria on the petioles provideprimary inoculum the next spring. Much of thisconidial production appears to be in late April andMay, coinciding with bud-break and new shoot growthof bur oak. Infection of newly emerged shootsapparently results in an endophytic or latent phase,with petiole necrosis and death of leaves occurringmonths later. Latent periods of several weeks alsowere seen when expanding shoots and young leaveswere inoculated without wounding in the greenhousestudies. A latent phase in the BOB disease cycle is notsurprising considering that Tubakia spp. are commonendophytes in Quercus spp. (Cohen 1999, Gonthier etal. 2006, Hashizume et al. 2008, Sieber 2007).

Conidia from crustose pycnothryia also may infectveins on the underside of fully expanded leaves in lateMay and June. Purple-brown discoloration on theunderside of the main and lateral leaf veins iscommon in June on trees with overwintering leaveswith crustose pycnothyria. The upper side of theseveins eventually become necrotic and produce pyc-nothyria with radiate scutella along the veins on thetop surface of the leaves. Naked sporodochia arecommon on the veins on the undersurface of theleaves. Such secondary inoculum may lead to sub-stantial disease buildup late in the season, especiallyduring wet summers, but many leaves infected late inthe season quickly develop vein necrosis and often fall

off the branches. In greenhouse inoculations of leafmidveins almost all inoculated leaves developed veinnecrosis and fell off the seedling after the necrosishad extended down to the petiole.

The LSU and ITS-rDNA sequences of T. iowensisare somewhat variable, more variable than is typicalfor an introduced, non-native ascomycete. The ITS-rDNA base substitutions found within T. iowensis arefound in isolates throughout the known geographicrange of the fungus, suggesting an establishedpopulation structure. However before the recentappearance of BOB Q. macrocarpa had been notedas a host of Tubakia spp. only from four herbariumspecimens collected in 1945 and the 1950s (Glaweand Crane 1989). The Wisconsin and Illinois speci-mens show leaf spots instead of vein necrosis typical ofBOB, and the leaf spots appear to be caused by a closerelative of T. iowensis, Tubakia sp. B, a speciescommonly found on Q. macrocarpa, Q. stellataWangenh. and Q. muehlenbergii Engelm.

Thus far BOB (vein necrosis and retention of killedleaves bearing petiole crustose pycnothryia) has beenfound only in Iowa and nearby states, especiallyeastern Nebraska and southern Minnesota. Bur oak isa widespread species covering much of eastern NorthAmerica (Deitschmann 1965), but the extent of BOBmay be coincidental with the geographic range of anorthern variety of bur oak, Q. macrocarpa var.oliviformis (F. Michx.) A. Gray, which has relativelysmall, olive-shaped acorns but is otherwise indistin-guishable from Q. macrocarpa var. macrocarpa (GreatPlains Flora Association 1986). Nixon et al. (1993) didnot recognize Q. macrocarpa var. oliviformis butinstead considered these savanna forests of the upperMidwest to be dominated by Q. macrocarpa 3 Q. albahybrids (5 Q. 3 bebbiana C.K. Schneid.). Suchhybrids may be common where the species overlap(Hardin 1975), but sites with BOB typically do nothave Q. alba and the northern variety Q. macrocarpavar. oliviformis appears to be a distinct taxon. It likelyhybridizes with var. macrocarpa where the two varietiesoverlap, but var. oliviformis tends to predominate onupland sites and var. macrocarpa on bottomland sites(Deitschmann 1965). Bur oak in eastern Nebraskawith smaller acorns are believed to be better adaptedto upland sites, while bur oak trees with larger acornsare thought better adapted to dense bottomlandforests (Laing 1966). We have seen severe BOBsymptoms only on bur oak with small acorns andmostly on upland sites, so adaptation of an ecotype ofvar. oliviformis to upland sites might be correlatedwith susceptibility to BOB.

The new fungus has been found throughout muchof Iowa, but we have observed more severe symptomsin the western two-thirds of the state, most commonly

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on upland sites with mature trees in open stands.Severe BOB also was found in hillier areas west of theMissouri River in Nebraska. Such upland savannaforests were once common in Iowa and neighboringstates before fire exclusion, and the preponderanceof these stately, open-grown relics of Q. macrocarpavar. oliviformis led to the designation of oak as Iowa’sstate tree. Fortunately there seems to be a widevariation in resistance to BOB, even in var. oliviformison upland sites where some trees in heavily diseasedgroves appear to be highly resistant. The dramaticsymptoms found initially suggested that the cause ofBOB was an introduced pathogen, but the apparentwide range in resistance among bur oak trees furthersuggests that T. iowensis and BOB are native to theregion.

In the past 20 y mature bur oak in Iowa haveexperienced more early season rainfall (NOAA 2011)and likely more disease pressure than in the previouscentury. The documented changes in Iowa’s climatehave included several factors that may favor BOB,including increased spring and summer precipitation,a shift from late season to early season precipitation,more summer humidity and warmer nighttimetemperatures (Takle 2011). The most critical pointin the BOB disease cycle appears be rain-splashdispersal of conidia from crustose pycnothyria andinfection of developing shoots in late April and May.Statewide nine of the past 10 y have received abovenormal precipitation during April and May comparedto the trend since 1895, and there has been nodrought since 1989 (NOAA 2011). Consecutive yearsof high spring rainfall may lead to accumulativebuildup of primary inoculum in a tree and depletionof its late-season energy reserves.

Many hardwood species in the western part of theMidwest appear to be poorly equipped to handle theadded disease pressure that comes with higher springand summer rainfall. With this change in climate(Takle 2011) there is a critical need for a betterunderstanding of the systematics and biology of foliarpathogens on native trees. Tubakia spp. on oaks andother hardwood species in eastern North America(Glawe and Crane 1987, Limber and Cash 1945,Tehon 1948) once were considered only minor leafspot pathogens (Munkvold and Neely 1990), but theyare now becoming more conspicuous and damagingand are in need of taxonomic reevaluation.

ACKNOWLEDGMENTS

The technical assistance of Sujin Kim, Kai Hillman and ZackGuinn is greatly appreciated. Numerous individuals provid-ed helpful material for study, including Donna Brandt,Christine Engelbrecht, Tivon Feeley, Aron Flickinger,

Otmar Holdenreider, Laura Jesse, Judy O’Mara, Jill Po-korny, Amy Rossman and Kyoko Scanlon. The work uponwhich this publication is based was financially supportedthrough grants awarded by the Northeastern Area State andPrivate Forestry, U.S. Forest Service.

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