Gurjar Gayatri, Barve Maneesha, Giri Ashok, Gupta Vidya
Plant Molecular Biology Unit, Division of Biochemical Sciences, National Chemical Laboratory, Dr Homi Bhabha Road, Pune 411008, India.
Mycologia. 2009 Jul-Aug;101(4):484-95. doi: 10.3852/08-085.
In this study we demonstrate the synergistic use of gene-specific markers, ITS-RFLP, ISSR and AFLP for distinguishing Indian F. oxysporum f. sp. ciceris races. We also report for the first time that F. oxysporum f. sp. ciceris race 3, a wilt pathogen of chickpea in India, is actually F. proliferatum based on phylogenetic analysis with EF-1alpha sequence data. F. oxysporum f. sp. ciceris races 1, 2 and 4 were easily distinguished from "race 3" (F. proliferatum) by PCR amplification with oligonucleotides designed from conserved regions of Hop78 transposon (Hop 78), cutinase (Cut), desaturase (Dst). F. oxysporum f. sp. ciceris race 4 was distinguished with the xylanase 3 (xyl3) gene by absence of amplification product only in this race. The Xyl3 amplified-DNA fragment isolated and sequenced from F. oxysporum f. sp. ciceris race 1 was similar to the F-xylanase (Xyl3) gene of F. oxysporum f. sp. lycopersici. A TELD motif, which is characteristic of the F-xylanases family, was detected within the deduced amino acid sequence of F. oxysporum f. sp. ciceris. Similarly the F. oxysporum f. sp. ciceris Hop78 DNA fragment, which identified "race 3" (F. proliferatum), was homologous to the Hop78 transposon of F. oxysporum f. sp. melonis, including the 100 amino acid conserved domain and the characteristic CCHC motif. The internal transcribed spacer region-restriction fragment length polymorphism (ITS-RFLP) approach along with intersimple sequence repeat (ISSR) method also differentiated "race 3" (F. proliferatum). Races 1 and 2 were identified by unique AFLP patterns. Sequence characterization of race-specific AFLP products revealed significant homologies of these sequences with metabolically important genes.
在本研究中，我们展示了基因特异性标记、ITS-RFLP、ISSR和AFLP协同用于区分印度尖孢镰刀菌鹰嘴豆专化型的生理小种。我们还首次报告，基于EF-1α序列数据的系统发育分析，印度鹰嘴豆枯萎病病原菌尖孢镰刀菌鹰嘴豆专化型生理小种3实际上是层出镰刀菌。利用从Hop78转座子（Hop 78）、角质酶（Cut）、去饱和酶（Dst）保守区域设计的寡核苷酸进行PCR扩增，尖孢镰刀菌鹰嘴豆专化型生理小种1、2和4很容易与“生理小种3”（层出镰刀菌）区分开来。尖孢镰刀菌鹰嘴豆专化型生理小种4仅通过木聚糖酶3（xyl3）基因无扩增产物而与其他生理小种区分开来。从尖孢镰刀菌鹰嘴豆专化型生理小种1中分离并测序的Xyl3扩增DNA片段与尖孢镰刀菌番茄专化型的F-木聚糖酶（Xyl3）基因相似。在尖孢镰刀菌鹰嘴豆专化型推导的氨基酸序列中检测到F-木聚糖酶家族特有的TELD基序。同样，鉴定“生理小种3”（层出镰刀菌）的尖孢镰刀菌鹰嘴豆专化型Hop78 DNA片段与尖孢镰刀菌甜瓜专化型的Hop78转座子同源，包括100个氨基酸的保守结构域和特征性CCHC基序。内转录间隔区-限制性片段长度多态性（ITS-RFLP）方法与简单序列重复区间（ISSR）方法也能区分“生理小种3”（层出镰刀菌）。生理小种1和2通过独特的AFLP图谱鉴定。生理小种特异性AFLP产物的序列特征显示，这些序列与代谢重要基因有显著同源性。
