1

Fig. 1. Map of Southern Marmara, Türkiye, showing study sites denoted by 1–18 (with the distribution area of Turkish pine shown in dark green).

Table 1. Geolocation of study sites where wood traps were established in Southern Marmara, Türkiye.
Study site Geographical position Elevation (m) Study site Geographical position Elevation (m)
1. Kireç 39°34’46.6”N
28°21’42.1”E 		508 10. Küçükkuyu 39°37’02.6”N
26°38’19.3”E 		557
2. Gökçedağ 39°37’29.5”N
28°46’12.2”E 		467 11. Yeniçam 39°35’43.2”N
26°31’35.2”E 		379
3. Balya 39°46’52.1”N
27°43’33.5”E 		448 12. Ayvacık 39°36’14.7”N
26°24’52.80”E 		269
4. Savaştepe 39°22’07.0”N
27°37’34.9”E 		275 13. Bayramiç 39°53’11.2”N
26°40’44.9”E 		204
5. Konakpınar 39°24’57.7”N
27°57’44.8”E 		442 14. Çınarlı 40°4’53.6”N
26°22’24.9”E 		43
6. Sınıdırgı 39°14’58.3”N
28°20’17.4”E 		321 15. Dişbudak 40°20’59.8”N
27°00’24.3”E 		133
7. İvrindi 39°38’26.2”N
27°27’25.7”E 		310 16. Gelibolu 40°17’45.3”N
26°31’11.8”E 		26
8. Edremit 39°40’15.3”N
27°06’43.5”E 		372 17. Eceabat 40°14’04.0”N
26°24’10.9”E 		107
9. Burhaniye 39°22’19.5”N
26°59’19.8”E 		311 18. Yenice 39°58’41.6”N
27°13’0.6”E 		380
Table 2. Representative fungal isolates of Ophiostomatoid fungi associated with Orthotomicus erosus on Pinus brutia during the current study.
Taxon Isolate name Study site GenBank accession no. Close match in BLAST Accessison of match Identity %
LSU ITS
Ceratocystis ips CZ27.52 18 - OM867517 Ophiostoma ips* OM468593 99.7
CZ51.1 1 - OM867518 O. ips OM468597 99.7
CZ24.21 15 - OM867519 O. ips OM468593 99.7
CZ30.21 4 - OM867520 O. ips OM468597 100
Graphilbum sp. CZ20.19 11 OM883868 - Graphilbum rectanglosporium OM514754 99.1
CZ30.6 4 OM883867 - G. rectanglosporium OM514754 99.1
CZ30.24 4 - - - - -
CZ44.9 17 - - - - -
Leptographium wingfieldii CZ33.40 7 - OM885001 Leptographium wingfieldii KP691916 99.7
CZ41.7 14 - OM885002 L. wing.fieldii KP691916 100
CZ40.25 13 - OM885003 L. wing.fieldii KP691916 100
CZ42.32 15 - OM885004 L. wing.fieldii KP691916 100
* Ophiostoma ips (syn. for Ceratocystis ips).
Table 3. List of reference sequences used for the phylogenetic tree in this study and their GenBank accesion.
Species name Isolate number Type1 Isolated from Country2 Collector GenBank accession numbers3
LSU ITS
Ceratocystiopsis manitobensis UM237 T P. resinosa/Manitoba beetle gallery CAN J. Reid EU913674
Graphilbum anningense CXY1939 P. yunnanensis/T. yunnanensis CHN H.M. Wang MH325162
G. cf. rectangulosporium VPRI43763 P. radiata AUS A.J. Carnegie MW046118
G. cf. rectangulosporium VPRI43843 P. taeda AUS C. Trollip MW046119
G. crescericum CMW22828 T P. radiata/H. palliatus ESP P. Romón OM514749
G. fragrans CBS 279.54 T SWE A. Mathiesen-Kaeaerik MH868872
G. fragrans CBS 279.54 P. sylvestris SWE T.C. Harrington AF198248
G. ipis-grandicollis VPRI43762 T Pinus radiata/I. grandicollis AUS A.J. Carnegie MW046117
G. rectangulosporium CMW26258 M. Procter OM514754
Grosmannia aurea CMW667 A Pinus contorta var. latifolia CAN R.W. Davidson OM501387
Leptographium longiclavatum SL-Kw1436 CAN AY816686
L. lundbergii CMW2190 T P. sylvestris NOR H. Roll-Hansen OM501432
L. pyrinum CMW509 T D. adjunctus USA K.R.W. Davidson J. OM501445
L. terebrantis CMW29841 T D. terebrantis USA S.J. Baras JF798477
L. wingfieldii CBS 645.89 E T. piniperda FRA M. Morelet AY935603
L. wingfieldii MB192 P. halepensis/T. destruens gallery ISR M. Dori-Bachash KP691916
L. wingfieldii CMW4741 P. densiflora JPN H. Masuya OM501461
L. wingfieldii CBS 648.89 E P. brutia GRC Mich.-Ska. AY935611
L. wingfieldii MCC 125 P. densiflora JPN M. Masuya AY935608
L. wingfieldii CMW2096 P. strobus /T. piniperda EUR M. Morelet AY553398
L. wingfieldii CMW2096 T FRA FIN AY553398
Ophiostoma bicolor CBS492.77 T Picea glauca CAN S.M. Alamouti DQ268604
O. floccosum CMW34182 T Wood SWE A. Mathiesen-Käärik KU184431
O. fuscum CMW23196 T Picea abies/P. chalcographus FIN Linnakoski HM031504
O. ips AK188 Ips acuminatus UKR K.V. Davydenko KU663983
O. ips CMW7075 T I. i̇nteger USA CT Rumbold AY546704
O. ips MB176 P. halepensis/O. erosus ISR Dori-Bachash KP691908
O. ips CMW6418 P. elliottii/O. erosus ZAF XD Zhou AY546702
O. ips S36.9 Pine wood PRT C.S. Vicente OM468593
O. ips S40.1a P. pinaster PRT C.S. Vicente OM468597
O. ips MCC 023 Tp beetle JPN H. Masuya AY194935
O. japonicum CMW2202 T I. typographus japonicus/Picea jezoensis JPN Y. Yamaoka OM501492
O. montium CMW15419 P. contorta USA B. Bentz OM501498
O. piceae C1087 T DEU Münch AF198226
O. pseudobicolor CFCC52683 T I. subelongatus/Larix gmelinii CHN Q. Lu MK748188
O. quercus CMW2467 T Quercus spp. FRA M. Morelet AY466626
O. rectangulosporium MAFF 238951 JPN N. Ohtaka AB235158
1 T = ex-type, E = ex-epitype, A = authentic isolate.
2 AUS: Australia, CAN: Canada, CHN: China, DEU: Germany, ESP: Spain, EUR: Europe, FIN: Finland, FRA: France, GRC: Greece, ISR: Israel, JPN: Japan, NOR: Norway, PRT: Portugal, SWE:    Sweden, UKR: Ukraine, USA: United States of America, ZAF: South Africa.
3 ITS: internal transcribed spacer, LSU: ribosomal large subunit.
2

Fig. 2. (a) Colony characteristics of Ceratocystis ips on 2% MEA, 25 ℃ for 10 d: (b) light microscopic micrographs; peritechia, ostium and ascospores of C. ips, (c) phylogenetic estimate, based on the ITS and 5.8S regions of the nuclear rRNA operon sequence analysis, showing potential phylogenetic relationships. The tree is rooted in Graphilbum fragrans. The tree was constructed with the MEGA program and evaluated using the bootstrap procedure (1000 replicates). Only bootstrap values >50% were provided. The analysis involved 20 nucleotide sequences. There were 1354 positions in the final dataset. The isolates obtained in this study are shown in bold.

3

Fig. 3. (a) Colony characteristics of Graphilbum sp. grown on 2% MEA, 25 ℃ for 10 d. (b) Based on the LSU sequence analysis, a phylogenetic estimate showed potential phylogenetic relationships. The tree was constructed with the MEGA program and evaluated using the bootstrap procedure (1000 replicates). Only bootstrap values >50% were provided. This analysis included 11 nucleotide sequences. There were 896 positions in the final dataset. The isolates obtained from this study were printed in bold.

4

Fig. 4. (a) Colony characteristics of Leptographium wingfieldii grown on 2% MEA, 25 ℃ for 10 d. (b) Light microscopic micrographs: conidiophores and conidia of L. wingfieldii, (c) Phylogenetic estimate based on the ITS and 5.8S regions of the nuclear rRNA operon sequence analysis, showing the potential phylogenetic relationships. The tree is rooted in O. quercus. The tree was constructed with the MEGA program and evaluated using the bootstrap procedure (1000 replicates). Only bootstrap values >50% were provided. This analysis involved 18 nucleotide sequences. There were 842 positions in the final dataset. The isolates obtained from this study were printed in bold.

Table 4. Fungal species detected from Orthotomicus erosus adults, larvae, pupa and gallery (number of fungal isolates frequency) collected at all study sites.
Fungal Species Study site/s Gallery Larvae/Pupae Beetle Total %
Ceratocystis ips 1,2,3,4,5,6,8,10,11,12,13,14,15,16,17,18 13 15 463 481 69
Graphilbum sp. 1,2,3,4,6,11,12,13,15,17 2 - 24 26 4
Leptographium wingfieldii 1,2,3,4,5,6,7,10,13,14,15 8 2 177 187 27
Table 5. Length (and standard error) of the lesion in phloem in branches of Turkish pine 4 months after inoculation with Ophiostomatoid spp.
Species Isolate no. Tree no. Mean lesion length (mm) Re-isolation %
1th tree 2nd tree Total
Ceratocystis ips CZ24.21 1b/2a 52 ± 27.1 15.8 ± 4.9 33.9a ± 26.5 95.5
CZ27.52 3c/4b 67 ± 11.5 43.4 ± 6.8 55.2bc ± 15.3 86.4
CZ51.1 5a/6c 55.8 ± 8.6 75.2 ± 30.5 65.5c ± 30.5 86.4
CZ30.21 7b/8b 50.4 ± 9.9 51.8 ± 10.7 51.1bc ± 9.7 95.5
Total 51.4 ± 24.2 91
Leptographium wingfieldii CZ33.40 9b/10a 63.8 ± 69.9 25 ± 11.1 44.4b ± 51.4 86.4
CZ41.7 11b/12c 48.2 ± 6.7 83.6 ± 22.9 65.9c ± 24.5 68.2
CZ40. 25 13a/14a 33.6 ± 20.2 35.2 ± 5.8 34.4b ± 14.0 31.8
CZ42.32 15a/16b 24 ± 15.1 44.6 ± 19.6 34.3b ± 19.8 68.2
Total 44.8 ± 32.3 64
Graphilbum sp. CZ20.19 17b/18a 43.6 ± 9.5 28.6 ± 12.2 36.1b ± 13.0 77.3
CZ30.6 19a/20a 40.6 ± 14.2 21.4 ± 5.3 31.0a ± 14.3 40.9
CZ30.24 21a/22a 30.6 ± 13.9 28.4 ± 13.8 29.5a ± 13.1 4.5
CZ44.9 23a/24a 31.8 ± 3.4 26.6 ± 9.3 29.2a ± 7.2 86.4
Total 31.5 ± 12.1 52
Control 25a – 32a 25.3a ± 7.7  
Lesion length data were transformed (log10) before analysis though non-transformed values were reported. The same letters within the columns are not significantly different from one another at p < 0.0001