Molecular Detection of Diaporthe phaseolorum and Phomopsis longicolla from Soybean Seeds

Abstract

Species-specific detection of Diaporthe phaseolorum and Phomopsis longicolla from soybean seeds was accomplished using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and TaqMan chemistry. To use these detection systems, fungal DNA was released from soybean seed coats using an ultrasonic processor to break the cells. DNA fragment lengths ranged from 200 to 1,200 base pairs (bp), with the majority of fragments <500 bp. Based on DNA sequences of the internal transcribed spacer (ITS) regions of ribosomal DNA, three TaqMan primer/probe sets were designed. Primer/probe set PL-5 amplified a 96-bp fragment within the ITS1 region of P. longicolla, D. phaseolorum var. caulivora, D. phaseolorum var. meridionalis, and D. phaseolorum var. sojae. Set PL-3 amplified a 86-bp DNA fragment within the ITS2 region of P. longicolla. Set DPC-3 amplified a 151-bp DNA fragment within the ITS2 region of D. phaseolorum var. caulivora. TaqMan primer/probe sets were able to detect as little as 0.15 fg (four copies) of plasmid DNA. When using PCR-RFLP for Diaporthe and Phomopsis detection, the sensitivity was as low as 100 pg of pure DNA. Among 13 soybean seed lots from Italy and the United States, the total Diaporthe and Phomopsis detected using a traditional seed-plating technique ranged from 0 to 32%. P. longicolla was most prevalent, followed by D. phaseolorum var. sojae. D. phaseolorum var. caulivora, which only occurred in 0.5% of the Italian seed lots, was not detected in the U.S. seed lots. D. phaseolorum var. meridionalis was not detected in either the U.S. or Italian seed lots. Using TaqMan primer/probe set PL-3, the frequency of P. longicolla was 18% in seed lot I3, similar to the frequency obtained from PCR-RFLP and potato dextrose agar plating detection. The frequencies of D. phaseolorum and P. longicolla in each seed lot obtained by the different detection methods were comparable with respect to total infection and individual species detection. However, TaqMan detection provided the fastest results of all the methods tested.

Molecular Characterization of Phomopsis vexans Isolates of Eggplant of Bangladesh

“…caulivora. Zhang et al (1998) also by using PCRrestriction fragment length polymorphism, detected genomic differences among D. phaseolorum var. caulivora isolates and D. phaseolorum var.…”

Phomopsis Seed Decay of Soybean

“…Prior to the pathogenicity tests, each isolate of P. longicolla was examined for sporulation, dimension of conidia, pattern of stroma, and presence or absence of hyaline, filiform, and hamate beta conidia and perithecia to confirm identification (Hartman et al, 1999). The identifications of soybean isolates were verified previously by sequence analysis of the ITS regions and the mitochondrial small subunit rRNA genes (Li et al, 2001;Zhang et al, 1998). The identities of 11 other Phomopsis spp.…”

“…These primers could be used to specifically distinguish PSD-causal pathogens from each other and from other soybean fungal pathogens (Zhang et al, 1997). There were no differences in ITS sequences among seven geographically diverse isolates of P. longicolla (Zhang et al, 1998). Results from analysis of DNA from the Diaporthe -Phomopsis complex using PCR-restriction fragment length polymorphisms (PCR-RFLP) and DNA sequencing in the ITS and the 5.8 ribosomal DNA, along with morphological examination, indicated that P. longicolla is a distinct species, that D. phaseolorum var.…”

“…meridionalis are varieties of D. phaseolorum, and that D. phaseolorum var. sojae may either be several varieties of D. phaseolorum, or possibly several distinct species (Zhang et al, 1998).…”

Developing of DNA-Marker to the Fusarium Oxysporum F. Sp. Lycopersici Resistance Genes of Tomato

“…In addition, the rapid development of new molecular techniques, combined with the increasing knowledge on structure and function of resistance genes, will help getting new molecular markers for MAS (Hulbert et al 2001). Such methods offer the advantage of reducing or eliminating the need for lengthy culturing and difficult morphological identification procedures (Hamelin et al 1993;Zhang et al 1999). The potential benefits of this technology have been especially recognized in the regulatory field, where time and accuracy of identifications are crucial (Goodwin and Annis 1991;Smith et al 1996).…”