1、418 Plant Disease / Vol. 86 No. 4 418 Species Delimitation and Host Specialization of Ceratocystis laricicola and C. polonica to Larch and Spruce T. C. Harrington, Department of Plant Pathology, Iowa State University, Ames 50011; N. V. Pashenova, Labora- tory of Microbiology, Sukachev Institute of F
2、orestry, Krasnoyarsk, Russia; D. L. McNew and J. Steimel, Depart- ment of Plant Pathology, Iowa State University; and M. Yu. Konstantinov, Laboratory of Microbiology, Sukachev Institute of Forestry, Krasnoyarsk, Russia Bark beetles (Coleoptera: Scolytidae) are among the most important killers of tre
3、es in the family Pinaceae, especially on Pinus, Picea and Larix (15). Bark beetles are intimately associated with a variety of fungi, most notably, members of the genus Ophiostoma and their associated anamor- phic species of Leptographium (57). Al- though species of Ceratocystis sensu stricto are ge
4、nerally associated with other insects, there are three species of Cerato- cystis associated with tree-killing bark beetles (12,21). Two of these Ceratocystis species, C. laricicola Redfern an autoclaved 3-cm-long twig of Pinus strobus with bark removed was placed in the recipient plate before additi
5、on of molten MEA. Spermatizing cultures were grown on MYEA (2.0% malt extract, 0.2% yeast extract, and 1.5% agar), which is more suited than MEA for conidium production. The spermatizing cultures were flooded with sterile, deionized water. Co- nidia, conidiophores, and mycelial frag- ments were loos
6、ened by lightly scraping with a spatula, and 1 or 2 ml of this sus- pension was added to each recipient cul- ture. The recipient plate was swirled slightly to disperse the inoculum. Plates were incubated at room temperature (20 to 23C) and lighting, and the recipient plates were inspected periodical
7、ly for perithecia and ascospore production, which was gen- erally evident within 2 weeks, but plates were observed periodically for up to 5 weeks. The MAT-2 testers were also paired with themselves, but no perithecia were noted in these self-pairings, except that self-pairings of C1226-3 produced ab
8、un- dant perithecia without necks. Single-ascospore progeny was recovered as described by Harrington and McNew (8,9) after first dispersing the ascospores in a light oil (Isopar M). Germination of as- cospores was assessed by streaking the dispersed ascospores across a MEA-coated microscope slide, i
9、ncubating at room tem- perature for 24 h, and examining the spores at 400. Morphology. Because of the reported difference in size of the perithecial bases in C. laricicola and C. polonica isolates from Japan (25), we measured perithecial bases on MEA and other media, and measure- ments were made at
10、200 or 400. Inoculations. Inoculations were pre- formed in the Krasnoyarsk Territory and Khakhassya (southern Siberia, Russia). Six trees of Picea obovata (Siberian spruce) and 12 trees of Larix sibirica (Siberian larch) of 25 to 30 cm diameter at breast height (dbh) were selected for inoculation. T
11、he L. sibirica trees were inoculated on 24 June 1998, and the P. obovata trees were inoculated on 29 June 1998. Three of the P. obovata trees were on a well-drained site, while three others were in a poorly drained area with excessive soil moisture. The L. sibirica trees had been subjected to natura
12、l defoliation by the Siberian moth (Dendrolimus superans sibiricus Tschetvr.). Three of the L. sibirica trees were not ap- parently defoliated, three of the trees had an estimated 50% foliage missing, three trees were 75% defoliated, and three trees were completely (100%) defoliated at the time of i
13、noculation. Wounds were made in the stems in seven rings spaced at 80 to 200 cm height above the ground. Three wounds were evenly spaced around the circumference at each height, and the three wounds at each height were staggered with the wounds of adjacent heights. The wounds were made by a cork bor
14、er (8 mm diameter) to the depth of the cambium/xylem area. Inocu- lum consisted of mycelium and spores at the advancing margins of MEA grown isolates taken with the cork borer. On each tree, the three wounds of the top-most ring were not inoculated (controls), the next three lower rings were inocula
15、ted with C. polonica isolates pln24/96, C708, and C787, respectively, and the lowest three rings were inoculated with C. laricicola isolates lr05/94, C746, and C178, respec- tively. The bark was removed 5 weeks after inoculation and the vertical extent (above and below the inoculation point) of necr
16、o- sis in the cambium region was measured. A three-way analysis of variance (ANOVA) was performed with host, fungal species, and stress as the variables (SAS Institute Inc., Cary, NC). For stress, the lesion size in the three P. obovata trees on the well-drained site was compared with that in the th
17、ree trees on the poorly drained site, and the lesion size in the six L. si- birica trees with little defoliation (0 or 50% defoliation categories) was compared with the lesion size of the six L. siberica trees with 75 or 100% defoliation. RESULTS MAT-2 Sequences. Parsimony analysis of the aligned 197 characters from the DNA sequence of a portion of the MAT-2 HMG box determined that 48 characters were informative, 47 were parsimony unin- formative, and 102 characters were con- stant. Two most parsimonious trees of 128
