Salehi M, Izadpanah K, Siampour M
Agricultural and Natural Resources Research Center of Fars, Zarghan, Iran.
Department of Plant Protection, College of Agriculture, Shiraz University, Shiraz, Iran.
Plant Dis. 2008 Apr;92(4):649. doi: 10.1094/PDIS-92-4-0649A.
During a survey in 2003, safflower plants (Carthamus tinctorius) with phyllody symptoms were observed in production fields in several districts of Fars and Yazd provinces in Iran. Affected plants showed floral virescence, phyllody, proliferation of axillary buds, and little leaf symptoms. Incidence of the disease was less than 10%. Direct and nested PCR assays were used to verify association of phytoplasma with the disease. Total DNA was extracted from fresh, fine roots of eight phyllody-affected safflower plants and one symptomless plant. With phytoplasma universal primer pair P1/P7 (5'-AAGAGTTTGATCCTGGCTCAGGATT-3'/5'-CGTCCTTCATCGGCTCTT-3'), target DNA fragments of approximately 1.8 kb were amplified by direct PCR from phyllody-affected plants and Iranian cabbage yellows (ICY) phytoplasma used as a positive control. Reamplification of P1/P7 products with 16S rRNA gene primer pair R16F2n/R16R2 (5'-GAAACGACTGCTAAGACTGG-3'/5'-TGACGGGCGGTGTGTACAAACCCCG-3') yielded fragments of the expected size (1.2 kb) from all eight diseased plants and the ICY-positive control. No products were amplified from the symptomless plant by either assay. R16F2n/R16R2 products were subjected to restriction fragment length polymorphism (RFLP) analysis by separate digestion with AluI, HaeIII, HhaI, HinfI, HpaII, MseI, RsaI, Sau3AI, or TaqI endonuclease. Comparison of resulting RFLP patterns with published patterns of other phytoplasmas (2) tentatively identified safflower phyllody (SP) phytoplasma as a member of clover proliferation group 16SrVI, subgroup C. HhaI digests also differentiated SP from ICY phytoplasma, a previously reported subgroup 16SrVI-A strain (3). After sequencing of the 16S rDNA fragment (GenBank Accession No. DQ88948), a BLAST search determined that SP phytoplasma shared closest homology with 16SrVI group members ('Candidatus Phytoplasma trifolii') and related strains (4). Furthermore, phylogenetic analysis of 16S rDNA sequences revealed SP phytoplasma to be most similar (99.7%) to brinjal little leaf (BLL) phytoplasma (GenBank Accession No. X83431). Analysis of putative restriction sites in 16S rRNA gene sequences revealed that SP and BLL shared identical restriction profiles and that both differed from the 'Ca. Phytoplasma trifolii' reference strain (GenBank Accession No. AY390261) because of the absence of a single HhaI site and the presence of an additional MseI site. Although safflower phyllody disease has been previously reported in Israel, the associated phytoplasma was classified as a strain of the aster yellows subgroup 16SrI-B (1). To our knowledge, this is the first report of safflower as a host of a 'Ca. Phytoplasma trifolii'-related strain. References: (1) M. Klein. Plant Dis. Rep. 54:735, 1970. (2) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:1037, 2004. (3) M. Salehi et al. Plant Dis. 91:625, 2007. (4) K. Wang and C. Hiruki, Phytopathology 91:546, 2001.
在2003年的一项调查中，在伊朗法尔斯省和亚兹德省的几个地区的生产田中观察到出现叶变症状的红花植株（Carthamus tinctorius）。受影响的植株表现出花变绿、叶变、腋芽增殖和小叶症状。该病的发病率低于10%。采用直接PCR和巢式PCR检测来验证植原体与该病的关联。从8株受叶变影响的红花植株和1株无症状植株的新鲜细根中提取总DNA。使用植原体通用引物对P1/P7（5'-AAGAGTTTGATCCTGGCTCAGGATT-3'/5'-CGTCCTTCATCGGCTCTT-3'），通过直接PCR从受叶变影响的植株和用作阳性对照的伊朗甘蓝黄化（ICY）植原体中扩增出约1.8 kb的目标DNA片段。用16S rRNA基因引物对R16F2n/R16R2（5'-GAAACGACTGCTAAGACTGG-3'/5'-TGACGGGCGGTGTGTACAAACCCCG-3'）对P1/P7产物进行再扩增，从所有8株患病植株和ICY阳性对照中获得了预期大小（1.2 kb）的片段。两种检测方法均未从无症状植株中扩增出产物。R16F2n/R16R2产物通过用AluI、HaeIII、HhaI、HinfI、HpaII、MseI、RsaI、Sau3AI或TaqI内切酶分别消化进行限制性片段长度多态性（RFLP）分析。将所得RFLP模式与其他植原体的已发表模式进行比较（2），初步确定红花叶变（SP）植原体为三叶草增殖组16SrVI、亚组C的成员。HhaI消化也将SP与ICY植原体区分开来，ICY植原体是先前报道的16SrVI-A亚组菌株（3）。在对16S rDNA片段进行测序（GenBank登录号：DQ88948）后，通过BLAST搜索确定SP植原体与16SrVI组成员（“Ca. Phytoplasma trifolii”）和相关菌株具有最密切的同源性（4）。此外，16S rDNA序列的系统发育分析表明，SP植原体与茄子小叶（BLL）植原体（GenBank登录号：X83431）最为相似（99.7%）。对16S rRNA基因序列中推定的限制性位点进行分析表明，SP和BLL具有相同的限制性图谱，并且由于缺少一个单一的HhaI位点和存在一个额外的MseI位点，两者均与“Ca. Phytoplasma trifolii”参考菌株（GenBank登录号：AY390261）不同。尽管以色列此前曾报道过红花叶变病，但其相关植原体被归类为紫菀黄化亚组16SrI-B的一个菌株（1）。据我们所知，这是关于红花作为“Ca. Phytoplasma trifolii”相关菌株宿主的首次报道。参考文献：（1）M. Klein。植物病害报告54：735，1970。（2）I.-M. Lee等人。国际系统与进化微生物学杂志54：1037，2004。（3）M. Salehi等人。植物病害91：625，2007。（4）K. Wang和C. Hiruki，植物病理学91：546，2001。
