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Beech leaf disease symptoms caused by newly recognized nematode subspecies Litylenchus crenatae mccannii (Anguinata) described from Fagus grandifolia in North America



Symptoms of beech leaf disease (BLD), first reported in Ohio in 2012, include interveinal greening, thickening and often chlorosis in leaves, canopy thinning and mortality. Nematodes from diseased leaves of American beech (Fagus grandifolia) sent by the Ohio Department of Agriculture to the USDA, Beltsville, MD in autumn 2017 were identified as the first recorded North American population of Litylenchus crenatae (Nematology, 21, 2019, 5), originally described from Japan. This and other populations from Ohio, Pennsylvania and the neighbouring province of Ontario, Canada showed some differences in morphometric averages among females compared to the Japanese population. Ribosomal DNA marker sequences were nearly identical to the population from Japan. A sequence for the COI marker was also generated, although it was not available from the Japanese population. The nematode was not encountered in Fagus crenata (its host in Japan) living among nematode‐infested Fagus grandifolia in the Holden Arboretum, nor has L. crenatae been reported in American beech in Japan. The morphological and host range differences in North American populations are nomenclaturally distinguished as L. crenatae mccannii ssp. n. from the population in Japan. Low‐temperature scanning electron microscopy (LT‐SEM) demonstrated five lip annules and a highly flexible cuticle. Females, juveniles and eggs were imaged within buds with a Hirox Digital microscope and an LT‐SEM. Nematodes swarmed to the tips of freshly cut beech buds, but explants could not be maintained. Inoculation of fresh nematodes from infested leaves or buds to buds or leaves of F. grandifolia seedlings resulted in BLD leaf symptoms. Injuring dormant buds prior to nematode application, in fall or spring, promoted the most reliable symptom expression. The biogeography and physiology of anguinid nematode leaf galling, and potential co‐factors and transmission are discussed.
Forest Pathology. 2020;00:e12580.  
 1 of 15
© 2020 Blackwell Verlag GmbH
  Revised:24Ja nuary2020 
  Accepted:27Januar y2020
DOI : 10.1111/efp .12580
Beech leaf disease symptoms caused by newly recognized
nematode subspecies Litylenchus crenatae mccannii (Anguinata)
described from Fagus grandifolia in North America
Lynn Kay Carta1| Zafar A. Handoo1| Shiguang Li1| Mihail Kantor1|
Gary Bauchan2| David McCann3| Colette K. Gabriel3| Qing Yu4| Sharon Reed5|
Jennifer Koch6| Danielle Martin7| David J. Burke8
1Mycology & Nematology Genetic Diversity
&Biolog yLaborator y,USDA-ARS,Belts ville,
2Soybea n Genom ics & Imp rovement
Laboratory, Electron Microscopy and
4Agriculture&A grifoodCanada,Ottawa
Research and Deve lopme nt Centre, Ott awa,
ON, Canada
5Ontario Forest Resear ch Institute, Ministr y
of Natura l Resources and Forestr y, Sault Ste.
Marie, ON, Canada
8HoldenA rboretum,Kirtland,OH,USA
LynnK.Carta,Mycolog y&Nematology
Genetic Diversity & Biology Laboratory,
USDA-ARS,Belts ville,MD20705-2350,
Symptoms of beech leaf disease (BLD), first reported in Ohio in 2012, include in-
terveinal greening, thickening and often chlorosis in leaves, canopy thinning and mor-
tality.NematodesfromdiseasedleavesofAmericanbeech(Fagus grandifolia) sent by
the OhioDepartment ofAgriculture tothe USDA, Beltsville, MD inautumn 2017
wereidentifiedasthefirstrecordedNorthAmericanpopulationofLitylenchus crena-
tae (Nematology, 21, 2019, 5), originally described from Japan. This and other popu-
lations from Ohio, Pennsylvania and the neighbouring province of Ontario, Canada
showed some differences in morphometric averages among females compared to
thepopulationfromJapan. A sequenceforthe COI markerwasalsogenerated,al-
though it was not available from the Japanese population. The nematode was not en-
countered in Fagus crenata(itshostinJapan)livingamongnematode-infestedFagus
grandifoliaintheHoldenArboretum,norhasL. crenataebeenreportedinAmerican
beech inJapan. The morphological and host range differences in North American
populations are nomenclaturally distinguished as L. crenatae mccannii ssp. n. from the
onstrated five lip annules and a highly flexible cuticle. Females, juveniles and eggs
swarmed to the tips of freshly cut beech buds, but explants could not be maintained.
Inoculation of fresh nematodes from infested leaves or buds to buds or leaves of
F. grandifolia seedlings resulted in BLD leaf symptoms. Injuring dormant buds prior
to nematode application, in fall or spring, promoted the most reliable symptom ex-
pression. The biogeography and physiology of anguinid nematode leaf galling, and
identification, morphometrics, new continent detection, new subspecies, symptom
transmission, taxonomy
2 of 15 
   CARTA eT Al.
Symptoms of beach leaf disease (BLD) were first detected in north-
ern Ohio, USA in 2012 and have since been found in northern
Pennsylvania, New York, Ontario, Canada (Ewing, Hausman, Pogacnik,
Slot,&Bonello,2018)and Connecticut.SomeAmericanbeechtrees,
Fagus grandifolia Ehrh. and European beech trees, Fagus sylvatica L.
Specimens were sent to the Nematology Department at Ohio State
University, and the Ohio Departmentof Agriculture, Reynoldsburg,
Ohio. The beech leaf disease symptoms included interveinal darken-
ing, some puckering, crinkling and irregularly thickened leaves (Figure
1). Mature forest trees exhibited thinned crowns and branch dieback.
The interveinal darkening was similar to eriophyid mite damage, but
those bud mites were not noticed in association with infested leaves.
scope, showed very small angular lesions within the darkened areas.
The appearance was similar to foliar nematode damage that appears
as larger angular lesions. BLD is associated with tree mortality within
Morphologically, these nematodess had a relatively large,
slender body length, short stylet length, more posterior vulva
and higher c′ value (tail length/anal body width) than most
Aphelenchoides (Shahina, 1996) and Bursaphelenchus taphrorychi
(Tomalak, Ma lewski, Gu, & Qian g, 2017) from European b eech.
Unlike Aphelenchoides or Bursaphelenchus, females also had a
small, narrow, weak median bulb and 6 lateral incisures. Upon fur-
ther inspection, it was determined to be the first population of
Litylenchus crenataeKanzakietal.(2019)fromthewesternhemi-
sphere, herein designated a new subspecies. Other populations
from Ohio, Pennsylvania and Ontario, Canada were also charac-
terizedwith molecular markers andmicroscopicanalysisof their
morphology. Nematodes stages within buds and leaves were ex-
aminedandimaged.InordertofulfilKoch'spostulates,L. crenatae
mccanniissp.n.wasusedtoinoculateotherwisehe althyAm erican
beec hs eedling si nO hio,USAandOnt ario,Ca nadagre enhouse st o
test whether BLD symptoms would result.
2.1 | Plant materials
County),USAbyan Ohio DepartmentofAgriculturenurseryinspector
fromailingAmericanbeech trees Fagus grandifolia Ehrh. and European
beech trees, Fagus sylvatica L. Other leaf specimens in 2018 were
sent from Kirtland, Ohio (Lake County), Potter County, Pennsylvania,
Ontario, Canada for morphological and molecular confirmation using
28Sand ITS 1,2 rDNAmarkers. Somespecimenswere dissectedfrom
leaves in water, measured and imaged using a microscope. Some leaves
1983) for 5–10 days at room temperature to reveal nematodes in leaves.
2.2 | Microscopy
Nematodes from leaves were imaged on an Olympus BX51 microscope
equipped with polarization optics and with a DP73 camera (Olympus
AmericaInc.). Measurements in micrometresweretakenwith the cali-
brated measuring tool in the imaging program cellSens ver 1.6 (Olympus
America Inc.). Fixed specimens were processed for permanent slides
withthe formalin-glycerine method (Golden, 1990) andimagedwith a
WildMPS48LeitzDMRB compound microscope (LeicaMicrosystems).
Measurements and morphometrics were calculated on an Excel spread-
sheet.TheLT-SEMprotocolofCarta, Bauchan, Hsu,andYuceer(2010)
wasusedemployingaHitachiS-4700 fieldemissionSEM (HitachiHigh
TechnologiesAmerica,Inc.)withaQuorum CryoPrepPP2000 (Quorum
Technologies Ltd.) cryotransfer system to observe nematodes isolated
2.3 | DNA Isolation, amplification,
sequencing, alignment
2.3.1 | DNA extraction
nematode in a 0.2 ml PCR tube containing 25 μl of extraction buffer
10 of them, prior to being transferred to each PCR tube, were im-
aged as vouchers for morphological and morphometrical analysis.
PCR amplification and DNA Sequencing: The 3.5 kb ribosomal
DNA(rDNA), ranging from near-full length 18S,internal transcribed
of the imaged specimens using recently modified procedures of Carta
and Li (2019). Cytochrome c oxidase I (COI) was amplified by PCR with
ersused for amplificationandsequencingarelistedin Table1.Each
25 µl PCR reaction was prepared with 2 µl of the extract and 23 µl of
thePCRmastermixcontaining0.625 U DreamTaq™Hot Start DNA
andthentreated with ExoSAP-ITreagent(Affymetrix,Inc) according
tothe manufacturer'sprotocol.DirectDNA sequencing forthe COI
 3 of 15
was performed bidirectionally with anABI BigDye Terminator v3.1
kitand in an ABI3730xlDNAAnalyzer(AppliedBiosystems) owned
rived from Litylenchussp.specimens, 104H78and104H82werede-
positedtoGenBankwithaccessionnumbers forrDNA(MK292137,
with thos e from the type p opulation from Mo rioka, Iwate Pref., Japa n
forGenBankaccession numbers LC383723forSSU,LC383724for
2.4 | Nematode plant inoculations
2.4.1 | Nematode collection and quantification
Nematodes were isolated from leaves collected with severe BLD
symptoms, and a modification of the “water soaking” isolation method
symptomsofBLDwerecut into 1-cm2 pieces and placed in a petri
dish containing 4% potato dextrose agar. Leaveswere than soaked
FIGURE 1 Leafsymptomsincludedarkenedgreenbands,chlorosisandnecrosis,Perry,OHFall2017(a)AmericanbeechFagus
grandifolia; (b) European beech, Fagus sylvatica images of David McCann
TABLE 1 PrimersusedforPCRandsequencing
Primers Direction Sequence (5′3′) Loci PCR Sequence Reference
18 S - C L- F3 FCT TGTCTCA AAGATTAAGCCATGCAT 18S √ √ Cart aandWick(2018)
18 S - C L- F2 FCTGTGATGCCCTTAGATGTCC 18S √ CartaandWick(2018)
IT S - C L- F 2 FATTACGTCCCTGCCCT TTGTA 18S √ CartaandWick(2018)
rDNA1.58S RACGAGCCGAGTGATCCACCG 5.8S √ Cherry,Szalanski,ToddandPowers(1997)
AB28 RATATGCTTAAGTTCAGCGGGT 28S √ Vrain,Wakarchuk,LevesqueandHamilton(1992)
28 S - C L- F 2 FCGACCCGTCTTGA AACAC 28S This study
4 of 15 
   CARTA eT Al.
nematodes was carefully collected with a pipettor and then centri-
fugedat1 252 gfor2 mintoconcentratethenematodes.Waterwas
then removed through pipetting and nematodes resuspended into
using a Sedgewick rafter at a magnification of4X, on an Olympus
BH-2 dissecting microscope. Quantification wasmade to standard-
izethe numberofnematodes usedforleaf andbudinoculation.The
nematodes remaining in the sterile water were used for leaf and bud
inoculation as described below. Some nematodes treated with ethanol
ing. Nematodes used for autumn tree inoculation were collected on
3 October 2018, and those used for spring bud inoculation were col-
2.4.2 | Tree leaf and bud inoculation
Three dif ferent types of plant inoculations were conducted; (a) new
leaf inoculation, (b) dormant bud inoculation prior to winter dormancy
and (c) dormant bud inoculation just prior to leaf out in spring. For
new leaf inoculation, mature, 1-m tall American beech trees (Fagus
grandifolia) that had been kept dormant in cold storage were placed
began to grow after 2 weeks and newly emerged, fully expanded
leaves were available for inoculation in early October 2018. Four
treatments were conducted on each of four trees: uninjured leaf,
injured leaf, injured leaf that was inoculated with 100 µl of a water
that was inoculated with 100 µl of a water suspension containing 80 0
nematodes (8,000/ml). Leaves were injured using a sterile dissecting
needle by making small holes in the leaf tissue and by scraping the
needle across the underside and upper side of each leaf. Leaves were
injured as this was found to produce the highest level of nematode
leafcolonizati oninprevi ouswork(Zhenetal.,2012).Afterleafi njur y,
thel eafwa sw rap ped ina11×21cmKi mwipe (Ki mbe rly-C lar k)w hich
was lightly moistened with sterile water to make it adhere to the leaf
surface. Leaves that received no nematodes had 100 µl of sterile
water added to the sur face of the leaf underneath the surrounding
Kimwipe.Theleafwasthanenclosed in asterile,plastic samplebag
to maintain moisture close to the surface of the leaf. Leaves receiving
nematodeshad 100µlofsterilewatercontainingeither400or 800
nematodesaddedtot hele afsurfacea sdes cr ibedabove.Af te rnem a-
todeap pl icati on,th el eafa ndKimwi pewereenc lo sedinaste rilesam-
ple bag to maintain leaf moisture. This was essential since nematodes
were then placed in a warm greenhouse with supplemental lighting.
Bags were kept on the tre ated leaves for 3 days to allow for nematod e
house with atemperature between 12–25°Cwith a12-hrday-night
cycle. Trees were monitored for 5 months until leaf senescence and
fall associated with the onset of plant dormancy.
uninjured bud, injured bud, uninjured bud that received a water
suspension containing 170 nematodes and 80 eggs (Table 2),
and an injured bud that received a water suspension containing
170nematodesand80eggs.Buds werewrappedwithapieceof
Kimwipe asdescribed above and wettedwithsterilewaterto in-
sure adherence to the bud surface. Then, 100 µl of sterile water
wasadded undertheKimwipe tothebud surface. The budsthat
did not receive nematodes received a sterile water control treat-
ment, but for buds receiving nematodes the sterile water suspen-
or sterile water application, buds were carefully wrapped with
parafilm to retain moisture against the bud surface (Figure 7).
Buds were injured with a sterile dissecting needle such that 6 small
holes were poked into each injured bud to facilitate nematode
entry. Trees were then placed in a cold greenhouse where they
withambientdaylight),dormantconditionsfor4 monthsprior to
moving plants intoaheatedgreenhouse (12–25°C)to break bud
dormancy and stimulate leaf emergence and grow th. Leaves were
then monitored for BLD as they developed.
Addit io na lbudinoculationswerealsoma deinApril2019using
two trees that had been dormant through winter. Due to low re-
covery of nematodes fromdormant budsinApril 2019,onlyone
bud on each tree could be inoculated with nematodes. Therefore
four total buds, two per tree, were treated as a part of this exper-
iment. On each tree, one bud was injured with a dissecting needle
as described above and then treated with sterile water as a no
nematode control, and a second was injured but treated with a
water suspension containing approximately 110 nematodes (see
Table 2). Buds were cover ed with a Kimwipe an d parafilm as a
part of the inoculation as described above. The trees were placed
inaheated greenhouse(12–25°C)tobreak bud dormancyunder
ambient light. Parafilm and Kimwipe were removed after3days,
and buds broke dormancy and leaves emerged 2 weeks after bud
inoculat ion. We monitore d leaves for sym ptoms of BLD as they
2.5 | DNA extraction and nematode detection post-
tree and branch type. Leaftissuewascollected usinga1-cmsterile
cork corer, and 1 leaf punch of BLD symptomatic tissue was extracted
per tree.DNA wasextracted usinga bead-beatingapproach where
tissue was t ransferre d into a 1.5-ml bea d beating tube w hich con-
tained 300mgof400 µM sterileglass beads(VWR)and 200 mg of
1 mm steril e glass beads (C hemglass). Ab out 750-µl of 2% cet yltri-
methyl-am monium bromid e (CTAB) was added to ea ch tube as the
extraction buffer, and samples were bead beaten using a Precellys
homogenizer(Bertin Technologies)for 80 s to lyse cells andrelease
 5 of 15
formextraction(Burke, Smemo,López-Gutiérrez,&DeForest,2012)
followed by precipitation in 20% polyethylene glycol 8,000 with
2. 5M NaC l.Pre cipit ate dDNAw assub sequentl ydrie dan ds usp end ed
nematode specific ITS primers to detect nematodes within leaf and
budsamples.Forwardprimer TW81 (GT TTCCGTAGGTGAACCTGC)
Subbotin, & Moens, 2003; Vovlas, Subbotin, Troccoli, Liebanas, &
Castillo,2008) wereusedfornematodeamplification. Amplification
sionof10 min at72°C (Esmaeili, Heydari,&Ye,2017).PCR product
was visually checked using 1%agaros egels and ethidium bromide
staining. PCR product positive to nematodes was purified using a
Inc.). Sequences were generated through the Life Sciences Core
Laboratories Center (Cornell University). Sequences were identi-
fied with the EMBL/GenBank /DDBJ database entries and the NCBI
Blast tool through GenBank (https ://
Morphological identification of nematodes: Leaves showing BLD
symptoms and nearby bud tissue were also collected and sent to
Nematodes were dissected from plant tissue and stained with acid
fuchsin (Byrd et al., 1983).
Litylenchus crenataeKanzakietal.(2019)mccanniissp.n.(Tables3‒5
3.1 | Description
Females have a nearly continuous,slightlyoffsetlip(Figures2a‒d,
body shape (Figures 2, 3a,b and 6c),the st ylet infemalesis5%of
there is a small, narrow, weak median bulb without an obvious valve.
Thevulval regioniskinked and irregular (Figure4c,d). Theanterior
valanaldistance( VAD).TheVADis2.8±0.3(2.3–3.3)timesthetail
length.Therectumisapproximately one quarter of thetaillength,
ally tapering, slender, conical tail with an asymmetrically pointed,
often mucronate extension. The dist al tail in immature (Figures 2i
3.1.1| Localities and hosts
Perry,OhioonleavesofAmericanbeechtreesFagus grandifolia Ehrh.
and European beech trees, Fagus sylvatica L. specimens collected
inSeptember 2017 and received between 9/12/17,and 05/18/18;
Fagus grandifoliaspecimensfromHoldenArboretum,Kirtland, OH
TABLE 2 Litylenchus crenatae mccannii ssp. n. Leaf and bud inoculation results based on results obtained through 6 May 2019
Sample ID Date Treat Control Injury + Control
Injury + 400
Nematodes Injury + 800 Nematodes Notes
Leaf Tree 1 10- 3 -18 Negative Negative Negative Negative See below
Leaf Tree 2 10- 3 -18 Negative Negative Negative Negative
Leaf Tree 3 10- 3 -18 Negative Negative Negative Negative
LeafTree4 10 - 3 -18 Negative Negative Negative Negative
Sample ID Date Treat Control Injur y + Control
170 Nematodes + 80
Injury + 170
Nematodes + 80 eggs
Bud Tree 1 10 - 3-18 Negative Negative Negative Not open (dead?)
Bud Tree 2 10 - 3-18 Negative Negative Negative Positive BLD
Bud Tree 3 10 - 3-18 Negative Negative Positive BLD Positive (slight)
BudTree4 10 - 3 -18 Negative Negative Negative Positive (slight)
Bud Tree 5 4-23 -19 NA Negative NA Positive BLD May 6 open
Bud Tree 6 4-23 -19 NA Negative NA Positive BLD May 6 open
Note: ForBudtreeinoculation1–4,redandyellowlabelreceivedabout140juvenilenematodes,30adultnematodesand80eggsperbudif
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TABLE 3 Morphometrics of live adult female Litylenchus crenatae mccannii ssp. n
L. c. mccannii
Perry OH
9-20 17
L. c. mccannii
Kirtland, OH
L. c. mccannii
Crawford, PA
9-20 18
L. c. mccannii
Potter, PA
L. c. mccannii
Ontario, Canada
n27 12 11 14 13
Body L µm 889±119(625–1084) 907.6±28.6(853.1–952.7) 739.8±178.9(562.0–1015.0) 788.8±95.2(564.4–891.2) 836±115(789–1109)
BodyWµm 14.2±1.0(12.1–16.1) 13.2±0.6(12.1–14.0) 16.2±2.4(15.0–22.0) 12.8±2.9(10.6–22.2) 14.8±2.2(13.6–15.9)
Stylet µm 9.2±0.5(8.4–10.3) 9.5±0.7(8.8–11.4) 9.3±1.2(7.5–11.0) 9.6±0.8(8.8–11.5) 9.5±0.7(8.210.4)
Phary nx L µm 193.5±35.7(126.3–244.2) 209.8±6.4(200.1–221.0) 141.7±36.7(100–195) 193.7±26.9(126.0–224.1) 176.6±12.7(151.2–198.9)
PUS µm 34.3±6.1(22.7–45.0) 22.9±5.8(13.7–33.7) 27.9±11.7(17.7–64.0) 36.9±9.4(29.7–54.1)
Tail L µm 54.3±6.1(39.8–64.4) 55.6±3.1(51.6–61.9) 43.7±11.3(33.0–62.0) 50.6±6.5(31.5–57.0) 50.6±5.5(42.3–56.6)
a63.0±10.0(43.8–76.8) 68.8±4.0(64.3–78.8) 46.3±13.6(31.2–67.7) 63.5±11.8(28.6–74.7) 61.4±9.8(40.9–73.4)
b4.7±0.7(3.8–6.6) 4.3±0.2(4.1–4.6) 5.4±0.6(4.8–6.8) 4.1±0.5(3.5–5.0) 4.8±0.3(4.0–5.3)
c16.4±1.5(13.3–20.1) 16.3±0.7(15.0–17.6) 16.8±1.4(15.0–18.8) 15.7±1.7(13.4–20.2) 16.6±1.7(12.6–19.6)
c′ 5.7±0.8(4.3–7.9) 6.0±0.5(5.5–7.2) 5.3±1.2(2.2–6.8) 5.9±0.9(4.5–6.5)
V% 76.6±1.4(73–79) 77.1±0.7(76–78) 77.7±0.07(76.5–78.4) 76.6±1.0(75.3–78.3) 80.2±6.6(75.2–86.9)
PUS/VA D% 27±8(22–50) 15 20.9 25.5±3.4(20.4–30.6)
PUS/BW 2.8±0.5(1.9–3.8) 1.8±0.5(1.1–2.7) 2.3±0.7(1.6–3.9) 2.5±0.5(1.8–2.9)
 7 of 15
TABLE 4 Morphometrics of fixed and live, adult female Litylenchus crenatae mccannii ssp. n
L. c. mccannii
Perry, OH,
Live, young
L. c. mccannii
Perry OH
Fixed, young
L. crenatae
Fixed, young
L. c. mccannii
N. America
Live, mature
L. crenatae
Fixed, mature
n27 10 10 50 10
Body L µm 889±119(625–1084) 823±61(750–947) 863±33(837–915) 740–908(625–1109) 816±32(758–870)
BodyWµm 14.2±1.0(12.1–16.1) 11.4±1.1(9.9–13.5) 12.3±0.9(11.0–13.5) 12 .8 –16. 2 (1 0 .6–16 .1) 22.9±2.6(18.4–27.7)
Stylet µm 9.2±0.5(8.4–10.3) 9.7±0.9(8.5–11.2) 8±0.4(7.4–8.5) 9.3–9.6(7.5–11.4) 10.6±0.5(9.9–11.3)
Styl conus µm 4.6±0.4(3.6–5.2) 3.1±0.2(2.8–3.5) 3.3±0.2(3.9–4.6)
Phary nx L µm 193.5±35.7(126.3–244.2) 152.6±16.2(133–186) 203±5.9(192–213) 142–210 (10 0–24 4) 123±6.7(110–131)
PUS µm 34.3±6.1(22.7–45.0) 32±3.4(29–39) 23–37(14–64) 68±7.4(57–81)
Tail L µm 54.3±6.1(39.8–64.4) 48.3±6.2(34.5–56.4) 55±3.8(50–63) 44–56(31.5–64.4) 33±2.3(30–36)
a63.0±10.0(43.8–76.8) 72.9±9.3(61–86) 67.5±5.8(60.7–74.4) 46–69(31–79) 35.9±3.4(30.2–41.1)
b4.7±0.7(3.8–6.6) 5.4±0.7(4.5–6.6) 5.3±0.6(4.5–6.3) 4.1–5.4(3.3–6.8) 6.6±0.4(6.1–7.6)
c16.4±1.5(13.3–20.1) 17.4±3.3(13–25) 15.7±0.7(14.4–16.7) 15.7–16.8(12.6–20.2) 24.5±1.9(18.5–25.1)
c′ 5.7±0.8(4.3–7.9) 6.0±1.0(4.3–7.9) 6.3±0.5(5.5–7.4) 5.3–6.0(2.2–7.9) 2.9±0.3(2.5–3.3)
V% 76.6±1.4(73–79) 76.9±1.2(75–79) 77.4±0.5(76.6–78.3) 77–80(73–87) 81.5±1.0(79.4–83.2)
PUS/VA D% 27±8(22–50) 22.9±2.1(20.2–25.9) 15–25 (20–50) 57.9±7(47–73)
PUS/BW 2.8±0.5(1.9–3.8) 2.6±0.4(2.2–3.5) 1.8–2. 8 (1.1–3.9) 3.5±0.4(2.8–4)
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8/27/18; (41°62′52.2x″N, 77°93′19.5x″W) Susquehannock State
Pymatuning Reservoir,Jamestown, Crawford County, PA 9/21/18;
Ontario Canada 11/2018).
3.1.2 | Specimen designation and deposition
Thirty-seven slides (T709t (holotype), T6960- 6973p (paratypes),
7108-7110p, 7113-7126p, 7182-7186p) with 260 females and 10
males and 89 juveniles from Perry, OH were deposited in the United
States DepartmentofAgricultureNematodeCollection (USDANC)
National Collection of Nematodes Ottawa.
3.2 | Diagnosis
The Litylenchus crenatae mccannii ssp. n. young female population
fixedandmountedinearlyautumnfromNor thAmericahadalonger
stylet[9.7±0.9µmn= 10(8.6–11.2)vs.8.0±0.4 (7.4–8.5)n= 10,
p< .001]µm andstyletconus[4.6(3.6–5.2) vs. 3.1 ±0.2 (2.8–3.5)
p<.001]µmthanthetypepopulationfromJapan.Thephar ynxwas
alsosignificantlyshorter(152.6±16.2vs.203±5.9µm,p < .001) in
immature females, although the “b” ratio was not dif ferent. However,
the phar ynx was longer in mature females of 3 populations (p < .001)
but not for t hose in Crawfo rd County (142 ± 36.7 vs. 123 ± 6.7)
Thepost-uterinesac in maturefemaleswas shorter (36.9 ± 9.4vs.
68±7.4,p < .001) than the Japanese population. The tail was shorter
in the fixed immature female populations (48.3 ± 6.2 vs. 55 ± 3.8
33±2.3 p < .01, and p < .001 for 3 other populations) which was
alsoreflectedindifferent c(16.8±1.4vs.24.5±1.9,p < .001) and
c′(5.3±1.2 vs.2.9±0.3,p < .001) ratios. The body width was nar-
p < .001). The male population from Perry, Ohio also had a longer
styl et [11.2 (10.6–12) vs. 10.2 (9.9–11)] µm and st ylet conus [4.8
(4.4–5.3)vs.3.6(3.5–4.3)]µm,anda widerbody[16.7(13.5–20.3)]
µm than the fixed type population from Japan.
3.3 | Remarks
The morp hological a nd host range di fferences in N orth Ame rican
populations are nomenclaturally distinguished as L. crenatae mccan-
nii ssp. n., after the plant pathologist who first observed the nema-
todes in BLD affected leaves. Live, mature males and females (Tables
1‒3)hadagreatdeal ofvariationwithoverlappingranges,butcer-
tain differences were noted when population averages were com-
pared. The degree of dimorphism between immature and mature
TABLE 5 Morphometrics of adult male Litylenchus crenatae mccannii ssp. n
L. c. mccannii
Kirtland, OH
Live 2-2018
L. c. mccannii
Crawford, PA
Live 9-2018
L. c. mccannii
Ontario, Canada
Live 5-2018
L. c. mccannii
Perry, OH
Fixed 11-2017
L. crenatae
Fixed 6-2017
n8 3 5 48
Body L µm 657±64(554–772) 586.3±73.3(502–635) 611.8±109.1(511.8–778.2) 548.0±16.7(534.5–566.7) 707±41(642–773)
BodyWµm 16.7±2.3(13.5–20.3) 15±0(15) 15.4±1.5(13.1–17.6) 15.1±2.5(12.1–16.7) 12.4±0.8(11.3–13.5)
Stylet µm 11.2±0.4(10.6–12.0) 10±0(10) 9.8±0.3(9.6–10.1) 11.1±0.5(10.5–11.4) 10.2±0.4(9.9–11.0)
Styl conus µm 4.8±0.3(4.4–5.3) 3.4±0.1(3.4–3.6) 3.6±0.3(3.5–4.3)
Phary nx L µm 143.2±11.8(124.9–159.7) 121.2±13.4(117.8–134.9) 113.9±5.0(108.5–118.1) 135±14(116–157)
Tail L µm 34.9±3.3(30.1–41.5) 33.3±3.9(29.0–36.7) 35.3±1.6(33.7–37.9) 34±2.6(30–38)
a40.0±7.8(31.1–57.3) 41.9±0.6(41.5–42.3) 45.6±2.6(41.1–47.8) 36.1±5.4(33.3–44.1) 57.2±4.7(48.9–61.9)
b4.6±0.4(4.1–5.3) 4.5±4.1(3.9–5.0) 4.8±0.2(4.6–4.9) 4.3±0.3(3.9–4.8)
c18.9±2.0(16.2–22.7) 19.1±1.9(17.0–22.7) 15.5±0. 2(15.3–15.9) 21.1±2.0(18.5–25.1)
c′ 3.2±0.2(2.9–3.5) 3.4±0.3(2.8–3.9) 3.6±0.4(3.0–4.1)
Spicule L µm 17.1±2.4(13.7–19.7) 15±0(15) 16.5±2.1(14.3–17.6) 16.3±1.4(14.9–17.6) 15.6±1.2(14.2–17.7)
Gubernaculm L µm 6.9±0.7(6.4–8.0) 6±1(5–7) 5.9±0.2(5.7–6.0) 5.3±0.8(4.3–6.1) 6.5±0.4(6.0–7.1)
 9 of 15
females seen in the population from Japan was less than that seen in
themultiple NorthAmericanpopulations,primarilyreflectedinthe
narrower body of mature females.
The young overwintering adults were primarily found in October
- November, and reproductive, mature females predominated in
spring through early autumn. Multiple nematode life stages, but not
males, were found within buds, and males were found within leaves
from spring through autumn. In Ontario, the young overwintering
adults were only found in leaves on the ground in the wintering
months, while mature adults could still be found in buds.
FIGURE 2 Light microscopy (LM) of Litylenchus crenatae mccanniissp.n.fixedfemalespecimens.(a–k),live,polarizedlightmicroscopy
(g)maturevulva,post-uterinesac;(h)immaturetail.(i)maturevulva,tail;( j,k)maletailswithspicules;(l)slenderfemaletailwithmucro,
FIGURE 3 Polarizedlightmicroscopy
(PLM) of live Litylenchus crenatae mccannii
ssp. n. (a) female; (b) male; (c) eggs; (d)
(a) (b)
(c) (d)
10 of 15 
   CARTA eT Al.
Nematodes did not sur vive on potato dex trose agar plates
with Rhizoctonia solani.Rinsed,surface-sterilizedleavesembed-
ded in either water agar or potato dextrose agar did not provide
any better yield of nematodes than strips of leaves in sterile water
from which thousands of nematodes per infested leaf could be
harvested over a few weeks. Twigs below infested buds were
cut and placed in Baermann funnels or dishes but no nematodes
3.4 | Bud associated nematodes
Litylenchus crenatae mccannii ssp. n. adults were inoculated to freshly
dissected beech bud tips embedded in moist water agar plates that
resulted in adult females swarming onto the bud tip (Figure 6a).
Dissection demonstrated that nematodes entered the excised bud
but did not develop.
Neither bud nor leaf explant s could be maintained on water
agar. Nematode females and eggs were exposed from within buds in
March 2019 and imaged with a Hirox (Figure 5b–d) microscope and
3.5 | Tree leaf and bud inoculation
Wefound that beechleaf inoculations failed to initiate anysymp-
tomsofBLDintrees after5months of growth(Table4).Although
some leaves developed browning or leaf margins over the course of
5 months, none developed interveinal darkening that is characteris-
ticofBLD. Wealsoobservednoleafmortality duringthe5-month
incubation until trees began to senesce and enter dormanc y. Bud
inoculation however was successful in initiating symptoms of BLD
inbuds wherenematodeswereapplied(Table4andFigure8). For
which were injured prior to nematode inoculation showed evidence
of BLD, while the fourth bud failed to open and died. Control buds
which were injured or uninjured and which did not receive nema-
tode inoculation all opened without signs of BLD. However, injured
FIGURE 4 Low-temperaturescanningelectronmicroscopyoffemalemorphology(a)facewithstyletopening(verticalarrow),amphid
young females; (i, j) mature females
(a) (b) (c)
(h) (i) (j)
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FIGURE 5 Litylenchus crenatae
mccannii ssp. n. on and in buds. (a) LM
image of adult females swarming on bud
infested beech bud from the Holden
(a) (b)
(c) (d)
FIGURE 6 Low-temperaturescanning
electron microscopy images of Litylenchus
crenatae mccannii ssp. n. on leaf sheath
at bud base. (a) females, juveniles, eggs
on bud sheath; (b) adult females on bud
sheath; (c) adult female on bud sheath
near leaf mesophyll
(a) (b)
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   CARTA eT Al.
buds, even those without nematode inoculation, tended to open
more slowl y than uninjur ed leaves. Of th e 4 buds which rece ived
nematodes but were not injured, only 1 developed symptoms of BLD
while the other 3 buds opened normally and leaves appeared unaf-
fected. For spring inoculated buds, both buds which were injured
and received nematodes developed BLD symptoms even after only
2 weeks of incubation. Both buds which did not receive nematodes
opened normally and were free of BLD symptoms.
3.6 | Nematode identification and sequence identity
Leaf tissue symptomatic for BLD was extracted from trees that underwent
budinoculation.DirectsequencingofPCR productandBlastmatching
returned matches to Litylenchus crenatae with 99% identity confirm-
ing the presence of the nematode in leaves developed from inoculated
dissected and stained with acid fuchsin (Byrd et al., 1983) and stained
Multiplesymptomaticleavesreceivedby the USDA-ARSMNGDBL
and Agriculture Canada national nematology laboratories yielded
many nematodes of one species only. In heavily infested areas where
nematode counts were highest in symptomatic leaves, a very small
number of nematodes were occasionally found on asymptomatic
leaves, but across all samples nematodes were not found in the vast
majority of asymptomatic leaves (S. Reed unpublished data).
FIGURE 7 Inoculation of Fagus
grandifolia seedlings with nematodes.
Bud inoculation for nematodes involved
and adding nematode suspension under
loosely wrapped in parafilm to retain
moisture and permit nematodes to
nematodes collected from field grown
trees. The “I” indicates the bud was
injured prior to inoculation
(a) (b)
FIGURE 8 Symptoms from inoculated
seedlings. (a) Bud tree two, injured with
tree three, no injury with nematode
added. 29 May 2019
(a) (b)
 13 of 15
Litylenchus nematodes with slender and obese morphs exhibit
phenotypic plasticity as they develop, as with other nematodes in
the family Anguinidae. Sclerotized, cuticular landmark structures
like anal and vulval openings were much more difficult to identify in
ing stylet was always distinct, and consistently larger in mature than
immature specimens.
AswithL. crenataeinJapan(Kanzakietal.,2019)andL. coprosma
in NZ (Zhao, Davies, Alexander,& Riley,2011),slender and obese
morphs coexisted during much of the year, but obese morphs pre-
dominate d in the late spring th rough autumn. A s in Japan, males
were found in leaves in late spring through autumn, but males
were not foun d within buds in No rth Americ a during Septem ber,
November, March, or June. Eggs were found in buds rather than
leaves during autumn months despite careful dissection and staining
of leaves. However, it was possible to find eggs in leaves during late
spring which hatched within leaves to produce many nematodes by
late summer.
The fine resolution COI marker generated for this population
employed the same primers designed for another phylogenetically
laboratory of the author of the description of L. crenatae, but slightly
different conditions. These included an especially thermostable
ported here that was not reported for the population from Japan.
Simple leaf inoculations with nematodes from leaves failed
to produce symptoms in Ohio greenhouse tests reported here.
However, a different experiment in Sault Ste. Marie, Ontario, Canada
with two leaves per sapling injured with a needle, treated with a
atodes as in Ohio, was saturated to runoff in two 2 ml doses over a
72-hrperiod. Inthis September 2018inoculationexperiment,four
out of the five surviving treated seedlings had BLD leaf symptoms at
the beginning of June 2019, while all control seedlings were free of
symptoms. Half of the initially treated seedlings were weakened by
mites in the growth chamber and succumbed to freezing tempera-
tures in a poly house. Leaves and buds of the surviving plant s may
havebeeninjuredby freezingtemperature andmites.Itispossible
that the nematodes could have entered buds in the larger inoculum
volume runoff during these ostensible leaf inoculations to produce
symptoms. Entry into buds was the most reliable route for successful
inoculation of nematodes, whether this occurred in the fall or the
spring. Nematodes could enter buds on their own, but symptoms
appeared more routinely with injur y. In nature this might be facili-
tated by some type of vectororfreezingandthawing.However,it
toms to develop since an early spring inoculation in the greenhouse
produced symptoms.
While foliar nematodes are fairly common, nematodes that
exist high in the tree canopy are not well-known, though endo-
phytic Aphelenchoides were recently discovered in poplar leaves
(Populus sp.) (Carta, Li, Skantar, & Newcombe, 2016). Nematodes
like Litylenchus crenatae mccannii ssp. n. within the nematode family
Anguini dae that are asso ciated with har dwood leaves havi ng sim-
ilar swollen, chlorotic-becoming-necrotic mesophyll tissue include
Litylenchus coprosma(N ew Zealand, Zha o et al., 2011), L. crenatae
(Japan,Kanzaki et al.,2019),Ditylenchus leptosoma(Korea,Geraert
& Choi, 1990) and Subanguina chilensis (Chile, Vovlas, Troccoli, &
Moreno, 20 00). Except for leaf symptoms in L. coprosma, the others
were described as galls, though they are not discrete like the seed
galls of Anguina or eriophyid mite galls. These other anguinid nem-
atode leaf gallers were implicitly assumed to be the cause of their
symptoms. Those plants were not very economically import ant nor
did those symptoms accompany serious mortality, so there was no
indication for the need to follow up with proof of nematode patho-
genicity. None of these nematodes associated with galls have been
reported to have a fundamental association with another pathogen
updates of all species).
The sponginess of galled por tions of the BLD leaves may result
from pectinases, similar to host leaves of the related anguinid nema-
tode Ditylenchus dipsaci (Myers, 1965). Physiological investigation of
superficially similar leaf galls of the related Ditylenchus gallaeformans
increased phenolics and carotenoids that counteract oxidative and
light damage. However, the Ditylenchus nematode gall originated in
the primordium, had a vascular connection, exhibited hypertrophied
and hyperplastic mesophyll and promoted indeterminate growth.
In contrast the simpler mite gall exhibited determinate grow th in
the epidermis (Ferreira et al., 2018). Therefore, the nematode gall
affected the host plant more profoundly and systemic ally than the
gall mite. Because there is likely to be a similar vascular connec-
tion for this related anguinid leaf galler, a Litylenchus metabolite or
even an endophytic microorganism could have a profound effect
on the plant. The nematode itself might be discovered to produce
a toxin like that recently discovered in entomoparasitic nematode
Steinernema carpocapsae(Luetal.,2017).
FIGURE 9 Acidfuchsinstains(a)
female and male nematodes from peeled
beech leaf mesophyll; (b) male nematodes
stained from leaf of experimentally
inoculated seedling, May 2019. (c) female
nematode from inoculated seedling bud
May 2019
(a) (b) (c)
14 of 15 
   CARTA eT Al.
Nematodes within the Anguinidae related to Litylenchus may
harbour toxic Rathayibacter spp. bacteria specific to their plant
hosts ( Anguina agrostis, A. funesta, A. tritici on monocots: Dorofeeva
et al., 2018 and Mesoanguina picridis on a dicot: Starodumova et al.,
2017),but this appears to bea population-specificevent among a
fewknown species (Murray et al.,2017). In any event, preliminary
evidence from subtractive leaf biome profiling by one of the authors
found no suggestion of any Rathayibacter associated with the dis-
ease. However, work is underway to better understand the nem-
atode and beech microbiomes. The microbiomes of the pinewood
nematode, and beetle vectors are expected to illuminate the patho-
Lack of symptomatic, naturally infested Fagus crenata leaves
near infested Fagus grandifolia in Nort h America sug gests F. c re n-
atamayberesistanttothepopulationfromNorth Americabutnot
symptoms from the Pacific rim, that is a likely region of endemic-
ity.TheinitialNorthAmericanlocalitiesinOhio,Pennsylvania, and
NewYork,USA,andOntario,Canadaneighbour LakeErie,atrade
hub from wh ich invasive spe cies such as em erald ash bor er (EAB)
originate d (Muirhead et al., 2 006). Like EAB, h uman transpor t of
wood may have distributed this probably invasive nematode. The
nematode may have arrived on this continent through an inverte-
brate vector, as is also suspected for Bursaphelenchus antoniae that
Certainly, windborne rain is a likely local means of disease transmis-
sion. Inver tebrates are as well. For instance, a predatory mite was
found entangled with nematodes, and we have collec ted various
mites and insects from leaf surfaces. Spider mites were numerous
in the summer in Ohio beech stands, and they can be windborne
for many miles. There are many potential invasive beetle vectors as-
sociated w ith beech (Mor rison, Sweeney, Hugh es, & Johns, 2017;
Rabagli a, Vandenberg, & A ccivatti, 20 09) (that may be present i n
the geographic regions where BLD occurs. Finding enough of any of
these invertebrates with nematodes takes time and directed effort
howeve r.
Birds are another possible vector, as with transport of Lyme
disease through ticks (Loss, Noden, Hamer, & Hamer, 2016).
Beech nuts are a critical component of the food chain for birds in
NortheasternandAppalachian forests. Theyarehighinthe can-
opy and difficult to harvest before the birds consume them. Since
these nematodes inhabit leaf buds they may also inhabit flower
buds. If so, birds might ingest nematodes and distribute them di-
rectly. They might carry mites, ticks or insects that carry nema-
todes as well.
vector of an elusive, hidden pathogen, it has had a consistent natural
and experimental association with disease symptoms to date.
Wethank DavidChitwood, retired MNGDBL Research Leader, for
early guidance and Joseph Mowery, ECMU, USDA-ARS, Beltsville,
Adam Hoke for a ssistance w ith labora tory work an d tree inocul a-
tions. Wethank Tracey OlsonandThomas Hall,PADepartmentof
Agriculture, Harrisburg, PAand Sarah Johnson, Wellsborough, PA
for samples. Mention of trade names or commercial products in this
publication is solely for the purpose of providing specific informa-
tion and does not imply recommendation or endorsement by the
vider and employer.MihailKantor was supported in partbyanap-
pointment to the Research Par ticipation Program at the Mycology
andNematology Genetic Diversity and Biology LaboratoryUSDA,
ARS,Nor theastArea,Beltsville,MD,administeredbytheOakRidge
Institute for Science and Educ ation through an interagency agree-
Lynn Kay Carta
Zafar A. Handoo
Mihail Kantor
Colette K. Gabriel
Sharon Reed
David J. Burke
Alves, M ., Pereira, A ., Vicente , C., Matos, P., Henri ques, J., Lop es, H.,
… Henriq ues, I. (2018). The r ole of bacteria i n Pine Wilt Dise ase:
Insights from microbiome analysis. FEMS Microbiology Ecology, 94,
Burke, D. J., Smemo, K . A., López-Gutiérrez, J. C., & D eForest, J. L.
(2012). Soil fungi influence the distribution of microbial func tional
groups that mediate forest greenhouse gas emissions. Soil Biology
and Biochemistry, 53, 112119. https ://
nique for clearing and staining plant tissue for detection of nema-
todes. Journal of Nematology, 14,142–143.
Cart a,L.K.,Bauchan,G.R.,Hsu,C.-Y.,&Yuceer,C.Y.(2010).Description
of Parasitorhabditis mississippii, n.sp. (Nemata: Rhabditida) from
Dendroctonus frontalisZimmermann(Coleoptera:Scolytidae).Journal
of Nematology, 42,46–54.
Cart a, L. K., & Li, S. (2019). PCR amplification of a long rDNA seg-
ment with one primer pair in agriculturally impor tant nematodes.
Journal of Nematology, 51 , e2019–e2026. https://
Cart a,L.K.,Li,S.,Skantar,A.M.,&Newcombe,G.(2016).Morphological
and Mole cular charac terization of t wo Aphelenchoides endophytic
in poplar leaves. Journal of Nematology, 48, 28–33. https ://doi.
org /10. 213 07/jofn em-2017-0 06
Cart a,L.K.,&Wick,R.L.(2018).FirstreportofBursaphelenchus antoniae
from Pinus strobus in the U.S. Journal of Nematology, 50, 473–478.
transcribed spacer region of Belonolaimus (Nemata: Belonolaimidae).
Journal of Nematology, 29, 23–29.
Dorofee va, L. V., Starodum ova, I. P., Krauzova, V. I., Pri syazhnaya, N.
V.,Vinokurova,N. G.,Lysanskaya,V.Y.,…Evtushenko, L. I.(2018).
Rathayibacter oskolensis sp. nov., a novel actinobacterium from
Androsace Koso-poljanskii Ovcz. (Primulaceae) endemic to the
Central Russian Upland. International Journal of Systematic and
Evolutionary Microbiology, 68,1442–1447.
 15 of 15
nematode, Nothotylenchus phoenixaen. sp. (Nematoda:Anguinidae)
associated with palm date trees and it s phylogenetic relations within
thefamily Anguinidae. Journal of Nematology, 49,268–275.https://jofnem-2017-072
Ewing, C. J., Hausman, C. E., Pogacnik, J., Slot, J., & Bonello, P. (2018).
Beech leaf disease: Anemerging forest epidemic.Forest Pathology,
Ferreira, B. G.,Oliveira, D. C.,Moreira, A. S.F.P.,Faria, A.P.,Guedes,
L. M., Fr anca, M. G . C., … Isaias , R. M. S. (2018). A ntioxidant m e-
tabolism in galls due to the extended phenotypes of the associated
organisms. PLoS ONE, 13,e0205364.https://
Geraert, E., & Choi, Y. E. (1990). Ditylenchus leptosoma sp. n. (Nematoda:
Tylenchida), a parasite of Carpinus leaves in Korea. Nematologia
Mediterranea, 18,27–31.
Golden,A. M. (1990). Preparation and mounting nematodesfor micro-
scopicobservation.InB.M. Zuckerman,W.F.Mai,&L.R.Krusberg
(Eds.), Plant nematology laboratory manual ( pp. 197205). Amh erst,
Kanzaki,N., & Futai,K. A.(2002). PCRprimersetfor determinationof
phylogenetic relationships of Bursaphelenchus species within the xy-
lophilus group. Nematology, 4,35–41.
Kanzaki, N., Ichihara, Y., Aikawa, T., Ekino, T., & Masuya, H. (2019).
Litylenchus crenataen. sp.(Tylenchomorpha:Anguinidae) a leafgall
nematodeparasitizingFagus crenata Blume. Nematology, 21, 5–22.
Loss, S.R., Noden,B.H.,Hamer,G.L.,& Hamer,S.A. (2016).A quanti-
tative synthesisof theroleofbirds in carryingticks and tick-borne
pathogensin North America. Oecologia, 182, 947–959. https://doi.
A., &Dillman,A. R. (2017).Activatedentomopathogenicnematode
infective juveniles release lethal venom proteins. PLoS Path, 13,
Morrison,A., Sweeney,J., Hughes,C., &Johns,R.C.(2017).Hitching a
ride: Fi rewood as a potenti al pathway for ran ge expansion of an ex otic
beechleaf-miningweevil,Orchestes fagi (Coleoptera: Curculionidae).
Canadian Entomologist, 149,129–137.
Muirhead, J. R.,Leung,B., Van Overdijk, C., Kelly, D. W., Nandakumar,
K., Mar chant, K. R ., & MacIsaa c, H. J. (200 6). Modelling l ocal and
long-distance dispersal of invasive emerald ash borer Agrilus pla-
nipennis (Coleopt era) in Nort h America . Diversity and Distributions,
A., Roge rs, E. E., & S ubbotin, S . A. (2017). Rathayibacter toxicus,
other Rathayibacter species inducing bacterial head blight of
grasses, and the potential for livestock poisoning s. Phytopatholog y,
Myers, R . F. (1965). Amylase, cellulase, invertase and pectinase in
several free-living, mycophagus, and plant-parasitic nematodes.
Nematologica, 11,441–448.
Anisandrus maiche Stark (Coleoptera: Curculionidae: Scolytinae) from
North America. Zootaxa, 2137, 23–28. https://
Fischer,1894(Nematoda:Aphelenchida) withsomenewrecords of
the group from Pakistan. Pakistan Journal of Nematology, 14, 1–3 2 .
sequenceofRathayibactersp.str ainVKMAc-2630isolatedfromleaf
gall induced by the knapweed nematode Mesoanguina picridis on
Acroptilon repens. Genome Announcements, 5,e00650-17.
TanhaMaafi, Z., Subbotin, S.A., &Moens, M.(2003). Molecular iden-
tification ofcyst-formingnematodes(Heteroderidae)fromIran and
aphylogenybased onITS-rDNAsequences.Nematology, 5, 99–111.
h t t p s : / / d o i . o r g / 1 0 . 1 1 6 3 / 1 5 6 8 5 4 1 0 2 7 6 5 2 1 6 7 3 1
Thomas , W. K. (2011). Mole cular techniqu es. In Internatio nal Seabed
Authority (Ed.), Marine benthic nematode molecular protocol hand-
book (Nematode barcoding). Technical Study: No. 7, ISA Technical
Study Se ries (pp. 22–37). Kings ton, Jamaica: I nternational S eabed
Authorit y.
Bursaphelenchus taphrorychi sp. n. (Nematoda: Parasitaphelenchidae),
the second Bursaphelenchus species from lar val gallerie s of the
beech bark beetle, Taphrorychus bicolor (Herbst.) (Coleoptera:
Curculionidae: Scolytinae), in European beech, Fagus sylvatica L.
Nematology, 19,1217–1235.
Vovlas, N., Subbotin, S. A., Troccoli, A., Liebanas, G., & Castillo, P.
(2008). Molecular phylogeny of the genus Rotylenchus (Nematoda,
Tylenchida) and description of a new species. Zoologica Scripta, 37,
52 1–5 3 7.
Vovlas, N., Troccoli, A., & Moreno, I . (2000). Subanguina chilensis sp.
n. (Nematoda: Anguinidae), a new leaf-gall nematode parasitizing
Nothophagus obliqua, in Chile. International Journal of Nematology, 10,
1–8 .
Vrain,T.C.,Wakarchuk,D.A.,Levesque, A. C., &Hamilton,R. I.(1992).
Xiphinema americanum group. Fundamental and applied Nematology,
port of Litylenchus coprosma on Coprosma robusta. Australasian Plant
Disease Notes, 12(1),17.https://doi .org/10.10 07/s13314-017- 0242-9
Zhao,Z.Q., Davies,K .A .,Alexander,B.,&Riley,I.T.(2011).Litylenchus
coprosmagen.n.,sp.n.(Anguinata),fromleavesonCoprosma repens
(Rubiaceae)inNewZealand.Nematology, 13,29–44.
tance to Aphelenchoides fragariae in Hosta cultivars. Plant Disease, 96,
1438–144 4.
How to cite this article:CartaLK,HandooZ A,LiS,etal.
nematode subspecies Litylenchus crenatae mccannii
(Anguinata)describedfromFagus grandifoliainNorthAmerica.
For Path. 2020;00:e12580. htt ps ://doi .org/10.1111 /efp.12 58 0
... Molecularly, Meloidogyne fallax can be distinguished from M. chitwoodi and other species by the ITS rRNA and COI gene sequence [13]. Description (modified after Carta et al. [19] and Handoo et al. [20]). Female: Females have a nearly continuous, slightly offset lip region with five annules, long and slender to semi-obese body shape. ...
... Molecular characterization: Ribosomal DNA marker sequences of Litylenchus crenatae mccannii ssp. were nearly identical to the population of Litylenchus crenatae from Japan [19]. The internal transcribed spacer (ITS) rDNA and 18 S rDNA sequences for Litylenchus crenatae from Japan are 99.7% and 99.9% similar, respectively, to Litylenchus crenatae mccannii from North America. ...
... µm; (ii) wider body (16.7 (13.5-20.3)) µm than the fixed type population from Japan [19]. Measurements (see Table 7). ...
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Plant-parasitic nematodes (PPNs) are important pests that cause an estimated ten billion dollars of crop loss each year in the United States and over 100 billion dollars globally. The Animal and Plant Health Inspection Service (APHIS) within the U.S. Department of Agriculture maintains and updates the U.S. Regulated Plant Pest list. Currently, the number of PPNs regulated by APHIS includes more than 60 different species. This review focuses on the top ten most economically important regulated and emerging plant-parasitic nematodes and summarizes the diagnostics of morphological and some molecular features for distinguishing them. These ten major previously described nematode species are associated with various economically important crops from around the world. This review also includes their current distribution in the U.S. and a brief historical background and updated systematic position of these species. The species included in this review include three PPNs considered by the U.S. Department of Agriculture as invasive invertebrates Globodera pallida, Globodera rostochiensis, and Heterodera glycines; four regulated PPNs, namely Bursaphelenchus xylophilus, Meloidogyne fallax, Ditylenchus dipsaci, and Pratylenchus fallax; and the three emerging PPNs Meloidogyne chitwoodi, Meloidogyne enterolobii, and Litylenchus crenatae mccannii.
... However, Brzeski (1963) suggested a synonymization of Gracilacus with Paratylenchus because the pro posed diagnostic characters were unreliable for defining the genera. Although some authors concurred with the synonymy (Brzeski, 1998;Ghaderi et al., 2016;Siddiqi and Goodey, 1964), others accepted Gracilacus as a valid genus (Abdel-Rahman and Maggenti, 1988;Brzeski, 1995;Doucet, 1994;Esser, 1992;Geraert, 1965;Raski, 1991;Shahina and Maqbool, 1993;Van den Berg and Buckley, 1993) or a subgenus of Paratylenchus (Siddiqi, 2000). In the book on Tylenchulidae, Ghaderi et al. (2016) recognized 117 species of Paratylenchus. ...
... In another study, Wang et al. (2016b) described P. guangzhouensis, a species with the stylet averaging 47 µm long and based on their ITS rRNA phylogeny, the authors showed that this species was clustered with those four species having a long stylet. Recently, Munawar et al. (2021), Singh et al. (2021), Clavero-Camacho et al. (2021) published comprehensive phylogenies of the genus Paratylenchus. These phylogenetic analyses did not support a justification of erection for the genus Gracilacus and this genus was considered as a synonym of Paratylenchus. ...
... Specimens were measured with an ocular micrometer on Leitz DMRB compound microscope. Nematodes were observed with the low-temperature scanning electron microscopy (LT-SEM) using the techniques described in Kantor et al. (2020) and Carta et al. (2020). ...
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The pin nematode, Paratylechus beltsvillensis n. sp. collected from rhizosphere soil of a Virginia pine tree (Pinus virginiana Mill) growing in Little Paint Branch Park, Beltsville, Prince George's County, Maryland, USA, is described and illustrated along with light and scanning electron photomicrographs. Females, males, and juveniles of this new species were recovered from soil samples using the sugar centrifugal flotation and Baermann funnel extraction methods. Morphologically, females are short, body length ranging from 245 to 267 μ m, stylet from 70 to 75 μ m long with anchor shaped knobs, vulva located at 70-73% and small vulval flap, spermatheca large, and ovoid filled with sperms. Lateral field with three incisures, of which the outer two are prominent. Tail slender, having a rounded tail terminus. Males without stylet and have a degenerated pharynx, spicules = 17-20 µm and gubernaculum = 5.0-5.5 µm. Both morphological observations and molecular analysis of ITS and partial 28S ribosomal RNA gene sequences indicated that the specimens collected from the soil at Beltsville Park from rhizosphere soil samples from Virginia pine represents a new pin nematode species.
... The presence of a new subspecies of nematode, Litylenchus crenatae ssp. mccannii (LCM), is considered a necessary condition for symptom development but it may not be sufficient to cause disease, because the nematodes used in the experiments by Carta et al. (2020) were extracted from symptomatic leaves. In previous work (Ewing et al., 2021) we found that such leaves also contained specific bacteria in the genera Wolbachia, Erwinia, Pseudomonas, and Paenibacillus, and one fungal species in the genus Paraphaeosphaeria, while LCM was found in both symptomatic and disease-free beech trees. ...
... While the exact BLD disease cycle has not been clearly defined, studies show that LCM overwinters in American beech buds as well as attached and detached leaves; live nematodes are found in leaves throughout the growing season with the greatest numbers found in late summer/early fall Reed et al., 2020). Since BLD symptoms are present at bud break and do not progress throughout the season (Fearer et al., 2022), this suggests that LCM causes symptoms prior to bud break, which Carta et al. (2020) confirmed in their study, and LCM migration into the leaf occurs sometime before September (Reed et al., 2020). Therefore, it is possible that higher lignin levels in local-naïve leaves are preventing LCM/pathogen infection prior to bud break. ...
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The ability to detect diseased trees before symptoms emerge is key in forest health management because it allows for more timely and targeted intervention. The objective of this study was to develop an in-field approach for early and rapid detection of beech leaf disease (BLD), an emerging disease of American beech trees, based on supervised classification models of leaf near-infrared (NIR) spectral profiles. To validate the effectiveness of the method we also utilized a qPCR-based protocol for the quantification of the newly identified foliar nematode identified as the putative causal agent of BLD, Litylenchus crenatae ssp. mccannii (LCM). NIR spectra were collected in May, July, and September of 2021 and analyzed using support vector machine and random forest algorithms. For the May and July datasets, the models accurately predicted pre-symptomatic leaves (highest testing accuracy = 100%), but also accurately discriminated the spectra based on geographic location (highest testing accuracy = 90%). Therefore, we could not conclude that spectral differences were due to pathogen presence alone. However, the September dataset removed location as a factor and the models accurately discriminated pre-symptomatic from naïve samples (highest testing accuracy = 95.9%). Five spectral bands (2,220, 2,400, 2,346, 1,750, and 1,424 nm), selected using variable selection models, were shared across all models, indicating consistency with respect to phytochemical induction by LCM infection of pre-symptomatic leaves. Our results demonstrate that this technique holds high promise as an in-field diagnostic tool for BLD.
... As some of these microfauna are used as biocontrol agents, especially nematodes (Grewal et al., 2005), their presence in stemflow could provide insights into the uniformity and efficacy of biocontrol applications (Ellsbury et al., 1996) or indicate the presence of natural biocontrol agents-as hypothesized in Magyar et al. (2021). Other microfauna can be disease agents in tree canopies where, again, nematodes figure prominently on this list (Carta et al., 2020;Qin et al., 2021). Thus, hypothetically, the presence or abundance of particular types or species of microfauna in stemflow waters may indicate an active (or the potential for) infection in the canopy above. ...
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As watersheds are complex systems that are difficult to directly study, the streams that drain them are often sampled to search for watershed “signals.” These signals include the presence and/or abundance of isotopes, types of sediment, organisms (including pathogens), chemical compounds associated with ephemeral biogeochemical processes or anthropogenic impacts, and so on. Just like watersheds can send signals via the streams that drain from them, we present a conceptual analysis that suggests plant canopies (equally complex and hard-to-study systems) may send similar signals via the precipitation that drains down their stems (stemflow). For large, tall, hard-to-access tree canopies, this portion of precipitation may be modest, often <2%; however, stemflow waters, like stream waters, scour a large drainage network which may allow stemflow to pick up various signals from various processes within and surrounding canopies. This paper discusses some of the signals that the canopy environment may impart to stemflow and their relevance to our understanding of vegetated ecosystems. Being a conceptual analysis, some examples have been observed; most are hypothetical. These include signals from on-canopy biogeochemical processes, seasonal epi-faunal activities, pathogenic impacts, and the physiological activities of the canopy itself. Given stemflow’s currently limited empirical hydrological, ecological and biogeochemical relevance to date (mostly due to its modest fraction in most forest water cycles), future work on the possible “signals in stemflow” may also motivate more natural scientists and, perhaps some applied researchers, to rigorously monitor this oft-ignored water flux.
... mccanni in infected leaves throughout the range of BLD . While this nematode must feed for BLD symptoms to occur, the potential role of additional pathogens, like bacteria, in symptom development remains under investigation Carta et al., 2020). The North American population of the nematode displays differences in both morphology and host range from Japanese populations of Litylenchus crenatae Kanzaki, Ichihara, Aikawa, Ekino, and Masuya, 2019, thereby distinguishing it as its own subspecies, L. crenatae ssp. ...
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Over the past century, beech bark disease has dramatically altered the composition and structure of stands containing American beech (Fagus grandifolia). Management of beech bark disease has focused on reducing beech thickets and identifying resistant trees in aftermath forests. Beech leaf disease is a recently detected invasive disease, now also affecting beech forests. In 2019, a plot network was established in central North America to examine the extent of beech leaf disease spread and the severity of effects on trees and forests. Here, data from this plot network was used to determine the extent to which American beech is exposed to beech leaf disease, beech scale (Cryptococcus fagisuga), and beech bark disease (C. fagisuga and Neonectria spp. complex) in forests surrounding the Great Lakes in southwestern Ontario, northeastern Ohio, western Pennsylvania, and western New York. Beech leaf disease and scale were found to be well established, not only among sites but also in the different canopy layers. Beech bark disease was present, but occurrence was low. Pest accumulation differed between overstory and sapling layers. Beech leaf disease was the primary pest of saplings while both beech leaf disease and beech scale dominated in the overstory. Forest composition was similar throughout the study range; American beech, sugar maple, red maple, and white ash were abundant and common in the sapling and seedling layer. Few invasive plants were evident among saplings and seedlings. Sugar maple and red maple will likely fill small canopy gaps following beech decline or mortality. Intervention should be considered in stands with potential for increasing tree diversity or promoting beech leaf disease-resistant American beech trees to the overstory. New strategies are needed for beech conservation along with research to determine the extent of beech leaf disease resistance in American beech.
This is a compilation of articles published in the Special Issue Systematics, Morphological, and Molecular Characterization of Economically Important Plant–Parasitic Nematodes: A Themed Issue in Honor of Dr. Gary Bauchan in Plants. It includes a series of original research (seven) and review articles (four) focused on plant-parasitic nematodes including two new species description, Pratylenchus dakotaensis n.sp. and Xiphinema malaka n. sp. Nematodes are one of the most important pests globally and can cause up to 14% loss of food crops. In total, nematodes cause over $100 billion in global crop damage annually. To date, only a few thousand PPN species have been described. Nematode identification has traditionally relied on morphological and anatomical characters using light microscopy and, in some cases, scanning electron microscopy (SCN). Lately, integrative studies combining molecular diagnosis with morphology and taxonomy are used to accurately identify and describe nematode species. Detailed analyses of morphological and molecular data have both significantly contributed to our overall understanding of the dynamic and complex nature of plant–nematode interactions. We are grateful to all the authors who submitted their work to be included in this special issue.
American beech trees are critical sources of food and shelter for wildlife in northeastern U.S. forests. Beech leaf disease is an emerging forest disease first discovered near Lake Erie by Cleveland Metroparks biologists in 2012. Plant scientists from the U.S. and Canada demonstrated through the use of Koch’s postulates that symptoms could be transmitted in the greenhouse by nematodes new to North America, providing a focus for further research. This rapidly advancing disease recently reached the Atlantic coast up through Maine and down to West Virginia. This chapter documents high resolution fluorescent and low-temperature scanning electron microscopic images of diseased leaves that show reduced chlorophyll in infested leaves, damage and distortion of internal and external leaf tissue, and describes mites entwined with nematodes that could possibly transmit the disease. Various images of symptomatic leaves and a discussion of possible microbial co-factors that may relate to increased virulence in North America are included.
Investigating the disturbance regimes of unharvested forests helps us understand their past, present, and future trajectory and gives us a model for forest management. It can also clarify the relative importance of small-scale gap dynamics versus more severe disturbances. Here we used tree rings to examine the recruitment patterns, growth dynamics, and disturbance chronologies of American beech (Fagus grandifolia), sugar maple (Acer saccharum), and eastern hemlock (Tsuga canadensis) in A.B. Williams Woods, an old-growth forest in Ohio, USA, over the past 250 years. We found that beech and sugar maple recruitment peaked around 1900 and continued through the 1900s, while hemlock recruitment peaked during 1825–1875, then declined and effectively ended in the early 1900s. Hemlock grew fastest during the 1800s according to ring width and basal area increment, while sugar maple ring width surpassed beech and hemlock in the 1900s. All three species showed a dramatic increase in growth from 1980 to 2010. Beech and sugar maple established regardless of canopy gaps, but 73% of hemlocks originated in gaps. In most decades, <10% of trees experienced gap recruitment or growth release, suggesting that ongoing, endogenous canopy mortality was the primary disturbance shaping this forest. However, a more severe forest-wide disturbance occurred during the 1980s–1990s when the scale insect causing beech bark disease was introduced, with greater than 30% of living trees showing growth releases in those decades. Another synchronous release occurred in the 1930s when blight-killed chestnuts were removed; 16% of trees showed releases. Both of these intermediate-severity disturbances involved human introduction of invasive species. Thus, we documented a natural disturbance regime of small-scale gap dynamics, punctuated by more severe anthropogenic disturbances in the twentieth century. These relatively frequent, intermediate-severity events probably mean that the forest’s current composition is non-equilibrial. Hemlock may continue to decline while beech maintains its dominance by constant regeneration in both gaps and shade, and by responding to disturbance with root suckering and growth pulses. The codominance of sugar maple may be relatively recent, and perhaps temporary, as we found little sugar maple recruitment before 1875 or after 1950, and three times fewer sugar maple saplings now than in the early 1900s. Despite being protected as a park, the development of this old-growth forest has been shaped more by disease-causing invasive species than natural disturbances over the past century. This result emphasizes the pervasiveness of human impacts even in communities we look to as examples of natural pattern and process.
When studying a forest disease, understanding the phenological relationship of the host tree and its pathogens is essential for identifying optimal management strategies to help prevent future spread of the disease. Since beech leaf disease (BLD) is a recently discovered disease, information about the general epidemiology and symptom phenology is largely unavailable. This study sought to answer questions related to symptom progression by conducting two observational studies on 10 trees from Cleveland Metroparks during the 2019 and 2020 growing seasons. BLD symptoms are characterized by two distinct leaf symptom types: dark green interveinal banding pattern or completely dark green and thickened leaf. Since there is evidence that the exotic nematode Litylenchus crenatae ssp. mccannii is associated with symptom development after direct inoculation into the buds, we hypothesized that symptoms would be apparent on the leaves at bud break. In our study, we visually confirmed the presence of both BLD leaf symptom types at bud break in naturally infected trees. Along with visual confirmation, a generalized linear mixed model (GLMM) showed that symptoms do not change throughout the growing season as time was not a significant variable when comparing symptoms across a growing season. Using both a Fisher's exact test and GLMM, we also determined that BLD leaf symptoms from a single leaf–bud pair do not progress in a specific or predictable pattern through subsequent growing seasons. These results formally validate the timing of BLD symptom expression and patterns of severity between years which will assist in furthering our understanding of the BLD pathosystem.
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Beech leaf disease (BLD) was first reported in 2012 in Lake County, Ohio on American beech trees (Fagus grandifolia Ehrh.). Since then, it spread across the Northeastern United States and has been reported from Ohio, Pennsylvania, New York, New Jersey, Connecticut, Rhode Island, Maine, West Virginia, and Ontario, Canada (Carta et al. 2020; Marra and LaMondia 2020; Reid et al. 2020). The symptoms of BLD are characterized by dark interveinal banding of leaves appearing soon after spring flush that become chlorotic and necrotic through autumn, resulting in canopy thinning in advanced stages, followed in some young trees by death. Litylenchus crenatae mccannii has similar morphological characteristics with subspecies Litylenchus crenatae (Kanzaki et al. 2019) reported on Fagus crenata from Japan. However, that beech species has not shown BLD symptoms or yielded any L. crenatae mccannii in North America. There are several morphological differences between the two nematode subspecies. The North American subspecies has a shorter post-uterine sac and narrower body width in mature females, shorter tail in immature females, longer tail in mature females, and longer stylet in males when compared to the Japanese subspecies (Carta et al. 2020). BLD symptoms were found on American beech trees in Prince William Forest Park, Prince William County, Virginia in June, 2021. The affected leaves contained females, males, and juveniles with morphometrics consistent with L. crenatae mccannii (Carta et al. 2020). The crude genomic DNA from a live single Litylenchus was prepared with freeze-thaw lysis (Carta and Li, 2019). The ITS PCR was performed by using the procedures and primer set, ITS-CL-F2 and 28S-CL-R described in the previous study (Carta and Li, 2020). The visualization, the cleanup and the direct DNA sequencing of the PCR products were performed by using the procedures described in the previous studies (Carta and Li, 2018 and 2019). Sequences were the same as the previous study (Carta et al. 2020) and submitted to GenBank as accessions MZ611855 and MZ611856. This represents the first report of BLD in Virginia. It is also approximately 300 miles south of the 2020 detection of BLD from New Cumberland, WV, and represents the southernmost detection of the disease and nematode in North America.
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Ribosomal DNA has been a reliable source of taxonomic and phylogenetic markers due to its high copy number in the genome and stable variation with few polymorphisms due to the homogenizing effect of concerted evolution. Typically specific regions are amplified through polymerase chain reaction (PCR) with multiple primer pairs that generate often incomplete and overlapping regions between adjacent segments of 18S, ITS1, 5.8S, ITS2, and 28S rDNA nucleotide sequences when combined in tandem. To improve the efficiency of this effort, a strategy for generating all these molecular sequences at once through PCR amplification of a large ribosomal 3.3 to 4.2 kb DNA target was developed using primer 18S-CL-F3 paired with D3B or a new alternative 28S PCR primer (28S-CL-R) and other well-positioned and ribosomal-specific sequencing primers (including novel primers 18S-CL-F7, 18S-CL-R6, 18S-CL-R7, 18S-CL-F8, 5.8S-CL-F1, 5.8S-CL-R1, 28S-CL-F1, 28S-CL-R3, 28S-CL-F3, 28S-CL-R1, and 28S-CL-F2). The D1 region between ITS2 and 28S boundaries and the flanking sequence between 18S and ITS1 boundaries were fully revealed in this large nucleotide segment. To demonstrate the value of this strategy, the long rDNA segment was amplified and directly sequenced in 17 agriculturally important nematodes from the Tylenchida, Aphelenchida, and Dorylaimida. The primers and their positions may be employed with traditional Sanger sequencing and with next-generation sequencing reagents and protocols.
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Juvenile, female and male nematodes were discovered in wood chips of white pine Pinus strobus from Ashley Falls, MA. Initial observations suggested these nematodes might be PWN, but closer morphological and molecular characterization proved otherwise. Comparison of measured features with those in the literature indicated this nematode population had some unique characteristics. The specimens were identified as Bursaphelenchus antoniae Penas et al., 2006 based on 18S rDNA molecular sequence vs only 95% similarity with PWN B. xylophilus . Compared to the previously described Portuguese population of B. antoniae , the sequences generated for the MA population were 98.3% similar in the ITS1, 2 rDNA and 99.9% similar for 28S rDNA. There was 99.2% similarity between the COI sequences of the US and Portuguese isolates of B. antoniae . This population has morphology consistent with that of Penas et al., 2006; however, the female tail on this MA pine population is mucronate and more attenuated than in B. antoniae from Portuguese P. pinaster found in association with Hylobius sp. Ecological associations of both populations of B. antoniae are discussed.
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Animal-induced galls are considered extended phenotypes of their inducers, and therefore plant morphogenesis and metabolism may vary according to the species of gall inducers. The alterations in vacuolar and apoplastic polyphenols, carotenoids, chlorophyll fluorescence rates, PSII quantum yield, and phospholipid peroxidation were studied in galls induced by Ditylenchus gallaeformans (Nematoda) on Miconia albicans and M. ibaguensis (Melastomataceae), and by an unidentified Eriophyidae (Acarina) on M. ibaguensis. The focus currently addressed is gall metabolism as the extended phenotype of the gall inducers, and the neglected determination of gall functionalities over host plant peculiarities. Galls induced by D. gallaeformans on M. albicans and by the Eriophyidae on M. ibaguensis have increased accumulation of apoplastic and vacuolar phenolics, which is related to the control of phospholipid peroxidation and photoprotection. The galls induced by D. gallaeformans on M. ibaguensis have higher carotenoid and vacuolar polyphenol contents, which are related to excessive sunlight energy dissipation as heat, and photoprotection. Accordingly, antioxidant strategies varied according to the gall-inducing species and to the host plant species. The distinctive investments in carotenoid and/or in polyphenol concentrations in the studied galls seemed to be peculiar mechanisms to maintain oxidative homeostasis. These mechanisms were determined both by the stimuli of the gall-inducing organism and by the intrinsic physiological features of the host plant species. Therefore, the roles of both associated organisms in host plant-galling organisms systems over gall metabolism is attested.
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A rod-shaped, non-endospore-forming and non-motile bacterium, strain DL-329T, was isolated from the above-ground part of a plant, Androsace koso-poljanskii Ovcz. (Primulaceae), at the the State Natural Reserve 'Belogorie', Russia. On the basis of 16S rRNA gene sequence comparisons, the strain clustered with members of the genus Rathayibacter, showing the highest sequence similarity to Rathayibacter tritici (98.89 %), Rathayibacter rathayi (98.82 %) and Rathayibacter festucae (98.82 %). The DNA hybridization experiments demonstrated that strain DL-329T represents a separate genomic species. The results of comparative studies of physiological and chemotaxonomic characteristics, including cell-wall sugar patterns, polar lipid profiles, and the matrix-assisted laser desorption/ionization time-of-flight mass spectra of bacterial cells, allowed clear differentiation of VKM Ac-2121T from the recognized Rathayibacter species at the phenotypic level. Based on the data obtained, a new species, Rathayibacter oskolensis sp. nov., is proposed, with DL-329T(=VKM Ac-2121T=LMG 22542T) as the type strain.
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Nothotylenchus phoenixae n. sp. is described and illustrated from soil samples of palm trees in Kermanshah Province, western Iran. The new species is characterized by a body length of 784 (663 to 925) mm in females and 677 to 715 mm in males; a delicate stylet 6 (5 to 7) mm long and six lines in the lateral field; median bulb of pharynx fusiform, nonmuscular, and nonvalvate; isthmus elongate, slender ending to a pyriform basal pharyngeal bulb not overlapping intestine; postvulval uterine sac well developed, 15 (14 to 17) mm long, female tail elongate-conoid with pointed terminus; and male with adanal bursa and spicules 21 to 22 mm long (n = 2). The new species comes close in morphology and morphometrics to five known species of the genus, namely N. affinis, N. hexaglyphus, N. persicus, N. taylori, and N. uniformis. Molecular analyses of the partial 18S, D2/D3 expansion segments of the partial 28S and internal transcribed spacer (ITS) revealed this as a new species. The sequences of the partial 18S and 28S D2/D3 regions confirmed the close phylogenetic relationship between N. phoenixae n. sp. and other anguinids, but Nothotylenchus is clearly separated from Ditylenchus species and should be considered as a valid genus.
Anisandrus maiche Stark, an ambrosia beetle native to Asia, is reported for the first time in North America based on specimens from Pennsylvania, Ohio, and West Virginia. This is the twentieth species of exotic Xyleborina documented in North America. This species, along with three others occurring in North America, were formerly placed in Xyleborus Eichhoff, but currently are assigned to Anisandrus Ferrari. Descriptions of generic characters used to separate Anisandrus from Xyleborus, a re-description of the female A. maiche, and an illustrated key to the four North American species of Anisandrus are presented.
Beech leaf disease (BLD) is a currently undiagnosed and seemingly lethal disease that was discovered in 2012 on American beech trees (Fagus grandifolia) in north‐east Ohio in the United States. Since its discovery, BLD has spread rapidly and can now be found in forests in 10 counties in Ohio, eight counties in Pennsylvania and five counties in Ontario, Canada. The initial symptoms of the disease appear as a dark green, interveinal banding pattern on the lower canopy foliage. These initial symptoms typically occur in the shrub or sampling layer of a beech stand. The later symptoms result in solidly darkened leaves that are shrunken and crinkled. The symptoms appear to progress through the buds as the affected buds are eventually aborted and no new leaves are produced. We fear this disease has the potential to drastically alter the Eastern deciduous forests of the United States on its own and through potential compounding disease effects. In addition, BLD poses a threat to global forests as symptoms of the disease were detected on European (F. sylvatica) and Oriental (F. orientalis) beech species in nurseries in north‐eastern Ohio. Due to its rapid spread and variability in environmental conditions where it has been detected, it seems unlikely that BLD is an abiotic disorder. Thus, intense efforts are underway to determine the causal agent of BLD. Relevant stakeholders are advised to be alert for BLD symptoms in beech forests in the Northern Hemisphere, and substantial resources should be invested in understanding this emerging forest disease.
Litylenchus crenatae n. sp., isolated from leaf galls of Fagus crenata from Japan, is described and figured. The new species is characterised by its dimorphism in adult females, six (or more) lateral lines, a more or less pointed tail tip in both sexes, male bursa arising posteriorly and reaching to near tail tip, presence of a quadricolumella and a post-uterine sac in females. Litylenchus crenatae n. sp. is distinguished from its only congener, L. coprosma , by the number of lateral lines, six or more vs four; the lip morphology, offset with very shallow constriction or dome-shaped without clear constriction vs clearly offset; tail tip morphology, more or less pointed vs blunt; and structure of the median bulb, weakly muscular with a clear valve vs not muscular with an obscure valve. The molecular phylogenetic analysis confirms that the new species is close to, but clearly different from, L. coprosma .
Pine Wilt Disease (PWD) has a significant impact on Eurasia pine forests. The microbiome of the nematode (the primary cause of the disease), its insect vector, and the host tree may be relevant for the disease mechanism. The aim of this study was to characterize these microbiomes, from three PWD-affected areas in Portugal, using Denaturing Gradient Gel Electrophoresis, 16S rRNA gene pyrosequencing, and a functional inference-based approach (PICRUSt). The bacterial community structure of the nematode was significantly different from the infected trees but closely related to the insect vector, supporting the hypothesis that nematode microbiome might be in part inherited from the insect. Sampling location influenced mostly the tree microbiome (P < 0.05). Genes related both with plant growth promotion and phytopathogenicity were predicted for the tree microbiome. Xenobiotic degradation functions were predicted in the nematode and insect microbiomes. Phytotoxin biosynthesis was also predicted for the nematode microbiome, supporting the theory of a direct contribution of the microbiome to tree wilting. This is the first study that simultaneously characterized the nematode, tree and insect-vector microbiomes, from the same affected areas and overall the results support the hypothesis that PWD microbiome plays an important role in the disease development.
Bursaphelenchus taphrorychi sp. n. is described from the bark of European beech, Fagus sylvatica. All propagative stages of the nematode are numerous in larval galleries of the beech bark beetle, Taphrorychus bicolor, while dauer juveniles are transmitted to new breeding trees under the elytra of adult beetles. The new species is characterised by the body length of 782 (717-858) μm in female and 638 (475-789) μm in male, moderately slender body (a = 35.0 (31.7-36.5) and 35.5 (31.4-37.1) in female and male, respectively), spicules 12.0-16.0 μm long, lateral fields with four incisures (i.e., three bands), and the arrangement of the seven male caudal papillae (i.e., a single precloacal ventromedian papilla (P1), one pair of adcloacal ventrosublateral papillae (P2), one postcloacal pair (P3) located at ca 60% of the tail length, posterior to the cloacal aperture, and one pair (P4) of subventral papillae of a similar size as the previous pair, but with somewhat sunken tips, located near base of bursa). In the number and arrangement of caudal papillae, stout and curved spicules with prominent rostrum and condylus, small vulval flap, body narrowed posterior to vulva, four incisures in the lateral fields, and long post-uterine sac, B. taphrorychi sp. n. shares most of the key morphological characters with members of the sexdentati-group. However, the newly described species is unique amongst Bursaphelenchus species of this group by the combination of shape of female tail, shape of spicules, and some other morphometric characters. The close relation of B. taphrorychi sp. n. with members of the sexdentati-group has been confirmed by DNA sequencing and phylogenetic analysis of the 28S rDNA region. The taxonomic separation of the new species is also confirmed by the unique molecular profile of the ITS region (ITS-RFLP). In laboratory rearing, B. taphrorychi sp. n. can develop and reproduce on Botrytis cinerea cultures.