Evaluation of Cultivar Resistance to Soybean Cyst Nematode with a Quantitative Polymerase Chain Reaction Assay.

Heterodera glycines, the soybean cyst nematode, is a major pathogen of soybean. Effective management of this pathogen is contingent on the use of resistant cultivars; thus, screening for resistant cultivars is essential. The purpose of this research was to develop a method to assess infection of soybean roots by H. glycines with real-time quantitative polymerase chain reaction (qPCR). This method will serve as a prelude to differentiation of resistance levels in soybean cultivars. A reproducible inoculation method was developed by means of a sand column to provide active second-stage juveniles (J2). Two-day-old soybean roots were infested with 0 or 1,000 J2/ml distilled water per seedling. Twenty-four hours after infestation, the roots were surface-sterilized and genomic DNA (gDNA) was extracted. For the qPCR assay, a primer pair for the single copy gene HgSNO, which codes for a protein involved in the production of vitamin B6, was selected for H. glycines gDNA amplification within soybean roots. Compatible 'Lee 74', incompatible 'Peking', and cultivars with different levels of resistance to H. glycines were infested with 0 or 1,000 J2/ml distilled water per seedling. Twenty-four hours postinfestation, infected seedlings were transplanted into pasteurized soil. Subsequently, they were harvested at 1, 7, 10, 14, and 21 days postinfestation for gDNA extraction. With the qPCR assay, the time needed to differentiate highly resistant cultivars from the rest was reduced. Quantification of H. glycines infection by traditional means (numbers of females produced in 30 days) is a time-consuming practice. This qPCR assay has the potential to replace the traditional Female Index-based screening and improve precision in determining infection levels.

Assessment of Parasitic Activity of Fusarium Strains Obtained from a Heterodera schachtii-Suppressive Soil.

This study assessed the potential impact of various Fusarium strains on the population development of sugarbeet cyst nematodes. Fungi were isolated from cysts or eggs of Heterodera schachtii Schmidt that were obtained from a field suppressive to that nematode. Twenty-six strains of Fusarium spp. were subjected to a phylogenic analysis of their rRNA-ITS nucleotide sequences. Seven genetically distinct Fusarium strains were evaluated for their ability to influence population development of H. schachtii and crop performance in greenhouse trials. Swiss chard (Beta vulgaris) seedlings were transplanted into fumigated field soil amended with a single fungal strain at 1,000 propagules/g soil. One week later, the soil was infested with 250 H. schachtii J2/100 cm(3) soil. Parasitized eggs were present in all seven Fusarium treatments at 1,180 degree-days after fungal infestation. The percentage of parasitism ranged from 17 to 34%. Although the most efficacious F. oxysporum strain 471 produced as many parasitized eggs as occurred in the original suppressive soil, none of the Fusarium strains reduced the population density of H. schachtii compared to the conducive check. This supports prior results that Fusarium spp. were not the primary cause of the population suppression of sugarbeet cyst nematodes at this location.

Identification of Fungal rDNA Associated with Soil Suppressiveness Against Heterodera schachtii Using Oligonucleotide Fingerprinting.

ABSTRACT To understand the nature of a soil with suppressiveness against Heterodera schachtii, an rDNA analysis was used to identify fungi associated with H. schachtii cysts obtained from soils possessing various levels of suppressiveness. Because H. schachtii cysts isolated from these suppressive soils can transfer this beneficial property to nonsuppressive soils, analysis of the microorganisms associated with the cysts should lead to the identification of the causal organisms. Five soil treatments, generated by mixing different amounts of suppressive and fumigation-induced nonsuppressive soils, were infested with second-stage juveniles of H. schachtii and cropped with mustard-greens. Fungi were identified through an rDNA analysis termed oligonucleotide fingerprinting of ribosomal RNA genes (OFRG). Cysts obtained from soil mixtures consisting of 10 and 100% suppressive soil predominantly contained fungal rDNA with high sequence identity to Dactylella oviparasitica. The dominant fungal rDNA in the cysts isolated from the soil mixtures composed of 0.1 and 1% suppressive soil had high sequence identity to Fusarium oxysporum. Polymerase chain reaction (PCR) amplifications performed with sequence-selective primers corroborated the treatment-specific distribution of rDNA clones obtained by the OFRG analysis. When these sequence-selective PCR primers were used to examine H. schachtii cysts from biocidal soil treatments that produced various levels of suppressiveness, only the D. oviparasitica-like rDNA was consistently identified in the highly suppressive soils.

Bacterial rRNA genes associated with soil suppressiveness against the plant-parasitic nematode Heterodera schachtii.

The goal of this study was to identify bacteria involved in soil suppressiveness against the plant-parasitic nematode Heterodera schachtii. Since H. schachtii cysts isolated from the suppressive soil can transfer this beneficial property to nonsuppressive soils, analysis of the cyst-associated microorganisms should lead to the identification of the causal organisms. Our experimental approach was to identify bacterial rRNA genes (rDNA) associated with H. schachtii cysts obtained from soil mixtures with various levels of suppressiveness. We hypothesized that we would be able to identify bacteria involved in the suppressiveness by correlating population shifts with differing levels of suppressiveness. Soil treatments containing different amounts of suppressive and fumigation-induced nonsuppressive soils exhibited various levels of suppressiveness after two nematode generations. The 10%-suppressive-soil treatment contained numbers of eggs per gram of soil similar to those of the 100%-suppressive-soil treatment, indicating that the suppressive factor(s) had been transferred. Bacterial rDNA associated with H. schachtii cysts were identified using a culture-independent method termed oligonucleotide fingerprinting of rRNA genes. Bacteria from five major taxonomic groups (Actinobacteria, Cytophaga-Flexibacter-Bacteroides, alpha-Proteobacteria, beta-Proteobacteria, and gamma-Proteobacteria) were identified. Three bacterial rDNA groups contained clones that were more prevalent in the highly suppressive soil treatments than in the less suppressive treatments, indicating a potential involvement in the H. schachtii suppressiveness. When these three groups were examined with specific PCR analyses performed on H. schachtii cysts that developed in soils treated with three biocidal compounds, only one bacterial rDNA group with moderate to high sequence identity to rDNA from several Rhizobium species and uncultured alpha-proteobacterial clones was consistently associated with the highly suppressive treatments. A quantitative PCR analysis confirmed the association of this Rhizobium-like rDNA group with the H. schachtii suppressiveness.