Cultural and DNA-based identification of Sclerotinia trifoliorum infecting chickpea in the U.S.

Pisum Genetics

2007—Volume 39

Brief Communications

Cultural and DNA-based identification of
Sclerotinia trifoliorum infecting chickpea in the U.S.

Njambere, E.N.¹, Chen, W.², ¹Washington State Univ., Pullman, WA, U.S.A.

Frate, C.³, Temple, S.R.³, ²USDA-ARS, Pullman, WA, U.S.A.

Wu, B.M.³ and Muehlbauer, F.J.² ³Univ. of California-Davis, CA, U.S.A.

Summary

Sclerotinia trifoliorum is identified for the first time to be one of the causal agents of white mold of chickpea in
North America. Based on cultural characteristics, isolates exhibited 2 growth rates: fast growing and slow
growing. Fast growing isolates were identified as S. sclerotiorum and slow growing isolates as S. trifoliorum based
on ascospore morphlogy, and ability to induce pH change in growing media, presence and variation of group I
introns in the nuclear small subunit rDNA, and ITS sequences. Intraspecific differences were also evident among
isolates of S. trifoliorum. Both S. sclerotiorum and S. trifoliorum are pathogenic on chickpea.

Pisum Genetics

2007—Volume 39

Brief Communications

Sclerotinia trifoliorum Erikss. is speculated to be one of the causal agents of white mold of chickpea in
California (Njambere et al. 2006). Although three species of Sclerotinia (S. sclerotiorum, S. minor and S.
trifoliorum) have been reported to cause the disease in various parts of the world, S. trifoliorum has never been
reported infecting chickpea outside the Australia sub-continent. Identification of the three species has mainly
been based on morphological characteristics such as sclerotial characteristics and growth in culture medium
(Cother, 1977; Bretag and Mebald, 1987). More recently, scientists have used molecular tools to study members of
this group of fungal species (Holst-Jensen et al. 1998, Powers et al. 2001). Nevertheless, ascospore dimorphism is
still the most definitive character for identifying S. trifoliorum. Work was therefore undertaken to characterize
the Californian isolates based on cultural characteristics, ascospore dimorphism and PCR-based techniques. The
objective of this study was to develop reliable methods for identifying S. trifoliorum and to determine the
efficiency of using the methods for population studies.

Isolates used in this study were obtained from sclerotia or segments of diseased stems sampled from infected
chickpea plants from various locations in central California. Nine isolates were obtained. Four other previously
identified isolates of S. sclerotiorum from lentil (Lens culinaris Medik) and pea (Pisum sativum L.) and S.
trifoliorum from alfalfa (Medicago sativa) were also included in the study for comparison. To test for colony
growth rates, PDA plates were centrally inoculated with a 7-mm diameter disc and maintained at room
temperature (25°C) in the dark. Measurements were taken at 12 hours intervals until colonies covered the whole
plate. Measurements were also taken on a pH indicator media with bromophenol blue (50mg per L) modified
from Steadman et al. (1994). In order to observe ascospore morphology two methods were used to induce
carpogenic germination: the method with preconditioning developed for S. sclerotiorum (Cobb and Dillard, 2004)),
and the method without preconditioning developed for S. trifoliorum (Rehnstrom and Free, 1993). To determine
the presence and sizes of group 1 introns in the nuclear small subunit rDNA, total DNA was extracted from
sclerotia using the FastDNA® kit described by Chen et al. (1999). PCR amplifications were conducted using the
primers ITS1/ITS4 and ITS5/ITS4 for the ITS regions containing the 5.8S rDNA and using various combinations
of primers (NS5/NS6 and NS5/NS8), to amplify different regions of the small subunit rDNA. PCR products were
separated and product sizes estimated using 1% agarose gel electrophoresis along with standard DNA size
markers, to determine the presence of introns in the amplified DNA regions. The PCR products amplified with
primer pair ITS1 and ITS4 were purified for direct DNA sequencing. Nucleotide sequences were determined from
both strands using ABI PRISM 377 automatic sequencer (Applied Biosystems, USA) at the Sequencing Core
Facility of Washington State University. Sequence comparisons were carried out using BLASTn
(http://www.ncbi.nlm.nih.gov/BLAST).

Among the nine isolates two were identified as S. sclerotiorum and the rest were S. trifoliorum. Isolates
identified as S. sclerotiorum had fast growing rates and induced strong color change of the pH indicating medium,
whereas slow growing isolates induced no color change or very faint color change and were identified as S.
trifoliorum. Ascospore dimorphism was consistently observed among the slow growing isolates, but was not
observed in fast growing isolates. Slow growing isolates exhibited 4 large and 4 small ascospores in the asci,
whereas the fast growing isolates had all the 8 ascospores of similar size. All of the isolates of S. trifoliorum contain
the introns located at the conserved DNA sequence region between primers ITS 1 and NS5. Based on the type of
group I introns in the nuclear small subunit rDNA, intraspecific variation was evident among isolates of S.
trifoliorum. We detected two of the five subgroups identified by Powers et al. (2001) among such a small sample
size of nine isolates. The two base-pair differences in the ITS region between S. trifoliorum and S. sclerotiorum
reported by Holst-Jensen et al. (1998) are also confirmed in this report. Furthermore, the ascospore dimorphism,
ITS sequence variation, and group I introns in the small subunit rDNA correlated well with growth rates and
possibly oxalic acid production in culture. This has important implications for using the above methods in species
identifications for population studies.

Acknowledgments: We thank Tim Paulitz for providing an isolate of S. trifoliorum, and the National Sclerotinia Initiative for
financial support.



Pisum Genetics	2007—Volume 39	Brief Communications

1. Bretag, T.W., and Mebalds, M. I. 1987. Australian J. Exptl. Ag. 27: 141-148. 2. Chen, W., Gray, L.E., Kurle, J.E., and Grau, C.R. 1999. Mol. Ecol. 8:871-877. 3. Cobb, A.C., and Dillard, H. R. 2004. Plant Health Instructor. DOI: 10.1094/PHI-T-2004-0604-01. 4. Cother, E.J. 1977. Seed Sci. Tech. 5: 593-597. 5. Holst-Jensen, A., Vaage, M., Schumacher, T., and Johansen, S. 1999. Mol. Biol. Evolution 16: 114-126. 6. Njambere E., Chen, W., Frate, C., Temple, S.R., and Muehlbauer, F.J. 2006. Phytopath. 96: S85. 7. Power, K.S., Steadman, J.R., Higgins, B.S. and Powers, T.O. 2001. In: Young, C.S., Hughes, K.J.D. (eds.), Proceedings of the XI International Sclerotinia Workshop, Central Science Laboratory, York, England, pp 95-96. 8. Rehnstrom, A.L. and Free, S.J. 1993. Exptl. Mycol. 17: 236-239. 9. Steadman, J.R., Marcinkowska, J. and Rutledge, S. 1994. Canadian J.l of Plant Path. 16: 68-70.









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