Genetic resistance in soybean to Pythium damping-off and molecular characterization of Pythium populations on soybean in Arkansas

Pythium damping-off and root rot on soybean [ Glycine max (L.) Merr.] can cause poor stands and consequently lower yields and is found in all soybean-producing regions of the world. Pythium damping-off and root rot is generally controlled with fungicide seed treatments, but recently the cultivar Archer was reported to be more resistant to seedling diseases caused by Pythium spp. than 'Hutcheson'. In order to determine the inheritance of Pythium resistance in 'Archer', 'Archer' was crossed with 'Hutcheson' and the parents and F2:4 populations were screened in a growth chamber with P. aphanidermatum using a hypocotyl inoculation technique. F 2:4 lines from 'Archer' x 'Hutcheson' fit the model for a single dominant gene in 'Archer' that confers resistance to Pythium damping-off caused by P. aphanidermatum. Resistance to Pythium spp. was not linked to the Rps1k gene that confers resistance to Phytophthora sojae, as previously reported. The Pythium resistance gene was located 10.6 cM from Satt510 and 26.6 cM from Satt114 on the major linkage group F. The new resistance gene was tentatively named Rpa1. However, P. aphanidermatum is one of the seven Pythium species that can attack soybean roots. In order to characterize the diversity of Pythium species associated with soybean roots in Arkansas, field tests were performed at four locations and three different planting dates (mid-April, mid-May and mid-June) in Arkansas from 2002 to 2005. Field isolates were collected from soybean roots. The PCR-RFLP technique of the mitochondrially-encoded cytochrome oxidase II (cox II) gene was used to support morphological identification. Cox II gene is highly conserved within, but not across Pythium species, and thus has the potential for being used in species discrimination. DNA from 683 isolates recovered from soybean roots was amplified using mtDNA cox II gene specific primers for the genus Pythium. Cutting amplicons with three restriction enzymes (Alu I, Rsa I and Nla III) created 28 species-specific restriction fragment length polymorphism banding profiles that aid the accurate identification of isolates to species level. The 683 Pythium isolates belonged to 13 different Pythium species. The most frequent Pythium spp. recovered were Pythium sylvaticum (63%), followed by P. irregulare (13.5%), P. dissotocum (12.8%), P. ultimum var. ultimum (5.9%), P. ultimum var. sporangiiferum (1.4%), P. myriotylum (0.7%), P. torulosum (0.7%), P. oligandrum (0.6%), P. pulchrum (0.3%), P. spinosum (0.3%), P. acanthicum (0.2%), P. rostratum (0.2%), and P. heterothallicum (0.2%). Based on the PCR-RFLP results, for most Pythium species, intraspecific variation in the banding patterns was observed. The phylogenetic relationship of the Pythium spp. was determined by sequence alignment of the cox II gene. The species grouped into four major clades that were in general reflective of the sporangial morphology.;In order to determine the effect of planting date and the performance of soybean cultivars previously assessed as resistant and susceptible to Pythium under controlled environmental conditions, plant stand, yield, root and top weight, root discoloration and Pythium incidence was recorded. Overall results showed that early planting dates (April and May), when soil temperatures tend to be low to moderate, seemed to favor soybean agronomic characteristics, such as yield and plant stand in Arkansas; and later planting dates (June) favor root discoloration and Pythium incidence. These results correlate with the Pythium isolate recovery study, where there was a clear trend in higher Pythium spp, recovery with later planting dates. In general, Pioneer cultivars (94M70 and 94M41) that were assessed as resistant to Pythium spp. under controlled environmental conditions, were resistant to Pythium in regard to root discoloration and Pythium incidence, but not plant stand and yield. Pathogenicity tests of arbitrarily selected Pythium field isolates were performed. Out of 12 Pythium spp. evaluated, seven Pythium species were pathogenic to soybean (58%) (P. aphanidermatum, P. ultimum, P. torulosum, P. myriotylum, P. irregulare, P. sylvaticum and P. dissotocum ). P. aphanidermatum and P. myriotylum were pathogenic and highly aggressive at high temperatures. P. ultimum and P. torulosum were pathogenic and highly aggressive at low temperatures. P. irregulare was pathogenic at moderate temperatures. However, isolates of P. sylvaticum and P. dissotocum were moderate and weakly pathogenic to soybean at low and high temperatures, showing that these species could infect soybean under a wide range of environmental conditions.