Construction and characterization of two bacterial artificial chromosome libraries of pea (Pisum sativum L.) for the isolation of economically important genes.

Pea (Pisum sativum L.) has a genome of about 4 Gb that appears to share conserved synteny with model legumes having genomes of 0.2-0.4 Gb despite extensive intergenic expansion. Pea plant inventory (PI) accession 269818 has been used to introgress genetic diversity into the cultivated germplasm pool. The aim here was to develop pea bacterial artificial chromosome (BAC) libraries that would enable the isolation of genes involved in plant disease resistance or control of economically important traits. The BAC libraries encompassed about 3.2 haploid genome equivalents consisting of partially HindIII-digested DNA fragments with a mean size of 105 kb that were inserted in 1 of 2 vectors. The low-copy oriT-based T-DNA vector (pCLD04541) library contained 55 680 clones. The single-copy oriS-based vector (pIndigoBAC-5) library contained 65 280 clones. Colony hybridization of a universal chloroplast probe indicated that about 1% of clones in the libraries were of chloroplast origin. The presence of about 0.1% empty vectors was inferred by white/blue colony plate counts. The usefulness of the libraries was tested by 2 replicated methods. First, high-density filters were probed with low copy number sequences. Second, BAC plate-pool DNA was used successfully to PCR amplify 7 of 9 published pea resistance gene analogs (RGAs) and several other low copy number pea sequences. Individual BAC clones encoding specific sequences were identified. Therefore, the HindIII BAC libraries of pea, based on germplasm accession PI 269818, will be useful for the isolation of genes underlying disease resistance and other economically important traits.

First Report of Sclerotinia Stem Rot of Chickpea Caused by Sclerotinia sclerotiorum in North Dakota and Washington.

Chickpea (Cicer arietinum L.) is cultivated as a rotational crop in the cereal-based production system in the U.S. Pacific Northwest (PNW) and its production is expanding to other northern tier states. During July 2005, symptoms of Sclerotinia stem rot were observed on chickpea cv. Dwelley and Dylan in fields near Spangle, WA and Carrington, ND, respectively, with disease incidence of approximately ≤1% in affected areas at both locations. Symptoms included stem whitening, wilting, and stem breakage. Occasionally, white fluffy mycelium was observed; however, production of sclerotia on infected plants was rarely observed. Sclerotinia sclerotiorum was isolated from diseased stems collected from both states. The isolates produced a ring of sclerotia near the edge of potato dextrose agar (PDA) plates in 7 days and produced neither conidia nor other fruiting bodies in culture after 30 days. PCR amplification of the rDNA internal transcribed spacer region from two representative isolates and subsequent digestion with restriction enzymes, Mbo I and Taq I, produced identical banding patterns to previously identified isolates of S. sclerotiorum from pea from the PNW (2). Chickpea cvs. Dwelley and Spanish White (eight plants of each) were inoculated by fastening mycelial agar plugs from an actively growing colony on PDA onto the stems with Parafilm. Symptoms of stem whitening were observed as early as 2 days after inoculation, and the lesions extended upward and downward from the inoculation site. Wilting and stem breakage were also observed. Control inoculations of four plants of each cultivar with PDA plugs without mycelium produced no visible symptoms. S. sclerotiorum was consistently reisolated from inoculated plants but not from control plants. Chickpea had been grown in the PNW for more than 20 years without any reported incidence of Sclerotinia stem rot although the disease has been reported from Arizona (3) and Asian countries (1). This is likely because of the upright growth habit of the chickpea plant coupled with relatively dry conditions late in the growing season. Previous chickpea cultivars were very susceptible to Ascochyta blight, an early-season disease of chickpea in the PNW that reduced chickpea stands and canopy coverage. Current cultivars possess much improved resistance to Ascochyta blight, allowing greater vegetative growth to occur and creating microenvironmental conditions conducive to Sclerotinia stem rot. In North Dakota, where humid conditions prevail late in the growing season, symptoms of Sclerotinia stem rot had been observed in previous years but had not been documented because of a recent history of chickpea cultivation there. To our knowledge, this is the first report of confirmed Sclerotinia stem rot of chickpea in North Dakota and Washington. References: (1) G. J. Boland and R. Hall. Can. J. Plant Pathol. 16:93, 1994. (2) I. Jimenez-Hidalgo et al. Phytopathology (Abstr.) 94(suppl.):S47, 2004. (3) M. E. Matheron and M. Porchas. Plant Dis. 84:1250, 2000.