Identification of Rhizoctonia solani associated with field-grown tulips using ITS rDNA polymorphism and pectic zymograms

Abstract

Methods based on internal transcribed spacers (ITS) ribosomal DNA (rDNA) polymorphism and pectic zymograms (ZG) were compared for their use in routine identification of Rhizoctonia solani isolates occurring in flower bulb fields. Thirty three AG 2-t isolates, pathogenic to tulips, could be distinguished from AG 1-IC, AG 2-2IIIB and AG 2-2IV, AG 3 and AG 5 by means of ITS rDNA fragment length and after digestion with EcoR I from AG 4 and AG 5. AG 2-t isolates and two Japanese isolates, pathogenic to crucifers and tulips, had an estimated fragment size of 710 bp, whereas Dutch AG 2-1 isolates, non-pathogenic to tulips, showed an estimated fragment size of 705 bp on agarose gel. Digestion of AG 2-t and AG 2-1 isolates with EcoR I, Sau3A I, Hae III and Hinc II revealed four and five distinct ITS rDNA digestion patterns, respectively. In AG 2 isolates 2tR114, 21R14 and 21R61 a double digestion pattern, indicating different ITS sequences within an isolate, was found. The observed ITS fragment length polymorphism between isolates pathogenic and non-pathogenic to tulips were considered too small to be used in routine screening of field isolates. Sequencing of AG 2 isolates 21R01, 21R06, 2tR002 and 2tR144 showed a total ITS rDNA fragment length of 715, 713, 714, and 728 bp. As an alternative to ITS rDNA fragment length polymorphism, pectic enzyme patterns were studied using a commercially available vertical gel-electrophoresis system and non-denaturing polyacrylamide gels amended with pectin. Anastomosis tester isolates AG 1 to AG 11 revealed different ZG. Fifty AG 2-t isolates and five AG 2-1 isolates belonged to a homogeneous pectic zymogram group. We propose to assign AG 2 isolates pathogenic to crucifers and tulip to ZG5-1. AG 2-1 isolates, non-pathogenic to tulip, formed a heterogeneous group with 4 distinct ZG. Pectic zymography provides an easy, quick and unambiguous method for routine identification of large numbers of field isolates. Such a technique is needed for research on the dynamics of Rhizoctonia populations to develop environmentally friendly control measures of rhizoctonia disease in field-grown flower bulbs.