Abstract: This work is the first report on developing a method to detect and quantify the zearalenone-producing Fusarium species in foodstuff by real-time PCR assay with SYBR Green I. Based on the polyketide synthase gene (PKS4) sequences of four zearalenone-producing Fusarium strains, a specific primer set was designed and used to detect and quantify zearalenone-producing Fusarium in foodstuff. The system developed under study required only 100 mg infected maize flour for test, had ability to detect down to 10 copies of the target gene per reaction, and produced reliable quantitative data over three orders of magnitude. Compared with the conventional methods, it is more rapid, specific and sensitive to detect potential zearalenone contamination in food or feed production.
Abstract: PIRA-PCR (primer-introduced restriction analysis PCR) was developed to detect isolates of Fusarium graminearum with moderate resistance to carbendazim, a methyl benzimidazole carbamate (MBC)-group fungicide. Two primer pairs were designed and synthesized according to the nucleotide sequence of the β2-tubulin gene from F. graminearum. Fragments of 164 bp were amplified by nested PCR from isolates differing in carbendazim sensitivity. A HindIII restriction enzyme recognition site was introduced artificially by inner primers to detect a mutation at codon 167, and TaaI (Tsp4CI) restriction enzyme was used to detect a mutation at codon 200. The sensitivity of isolates to carbendazim was determined by analyzing electrophoresis patterns of the resulting PCR products after simultaneous digestion with both HindIII and TaaI. Results from PIRA-PCR and a conventional method (mycelial growth on agar) were identical but PIRA-PCR required only 7–8 h while the conventional method required 5–7 days. This study demonstrates that PIRA-PCR not only monitors the appearance of moderately resistant isolates, but can be useful for detecting genotypes of F. graminearum with moderate resistance to carbendazim.
Abstract:Orobanche ramosa is an important parasitic weed of several agriculturally important crops. A fungal strain identified as Fusarium oxysporum (named FT2) proved to be specific and highly virulent to O. ramosa plants and was proposed as a mycoherbicide for the biological control of this weed to be applied at the soil level. Considering that detecting and tracking the strain is of utmost importance to know the fate of the strain after its release, an amplified fragment length polymorphism (fAFLP) analysis was chosen and utilized with the aim to develop a molecular marker to specifically identify this strain. A wide population of F. oxysporum strains isolated from different hosts was screened against the mycoherbicide strain in order to identify specific fragments belonging to this strain. Two specific fragments were found and their DNA sequences were utilized for primer design. A primer pair (named FT2230F/FT2230R) proved to be strain–specific and it amplified a 232 bp DNA fragment of FT2. These primers were used to monitoring the presence of the F. oxysporum strain in the soil. Amplicons were detected from all the soil samples artificially infected by using known amounts of FT2 inoculum, whereas none of the primer sets amplified DNA from soils not infected by FT2.
Abstract:Fusarium oxysporum f. sp. cubense is the causal agent of Panama disease of banana. A rapid and reliable diagnosis is the foundation of integrated disease management practices in commodity crops. For this diagnostic purpose, we have developed a reliable molecular method to detect Foc race 4 isolates in Taiwan. By PCR amplification, the primer set Foc-1/Foc-2 derived from the sequence of a random primer OP-A02 amplified fragment produced a 242 bp size DNA fragment which was specific to Foc race 4. With the optimized PCR parameters, the molecular method was sensitive and could detect small quantities of Foc DNA as low as 10 pg in 50 to 2,000 ng host genomic DNA with high efficiency. We also demonstrated that by using our PCR assay with Foc-1/Foc-2 primer set, Foc race 4 could be easily distinguished from other Foc races 1 and 2, and separated other formae speciales of F. oxysporum.
Abstract: Contamination of cereals with mycotoxins produced by Fusarium is a worldwide problem requiring rapid and sensitive detection methods. This paper describes the development of a PCR protocol facilitating the detection of F. tricinctum, which belongs to the FHB (Fusarium Head Blight) complex responsible for contamination of cereal grains with enniatins and moniliformin. Sequence alignment of partial IGS rDNA revealed a single nucleotide polymorphism, which was used to design primers differentiating F. tricinctum from other members of the FHB complex. The specificity of the assay was tested on 68 isolates belonging to 21 Fusarium species originating from different parts of the world and hosts/substrates. Positive PCR results were obtained from all 12 F. tricinctum isolates tested; however, unexpected amplicons were amplified from the templates of F. acuminatum (CBS 618.87) and F. nurragi (CBS 393.96). No cross reactivity was found with any other Fusarium species tested. The PCR assay was tested on 24 asymptomatic wheat seed samples originating from Northern Poland and resulted in 13 positive samples, of which 11 samples were contaminated with moniliformin and/or antibiotic Y.
Abstract: A test system for the diagnostics and identification of seven toxigenic fungi causing Fusarium head blight (Fusarium graminearum, F. culmorum, F. poae, F. sporotrichioides, F. langsethiae, F. avenaceum, and F. tricinctum) was developed using PCR. The identification of pathogen is based on the specific amplification of a DNA fragment of the gene of translation elongation factor 1 alpha (tef-1α) and subsequent detection of the results by the fluorescent amplification-based specific hybridization method. The system was tested on 38 isolates of different fungi of the genus Fusarium.
Abstract:Phytophthora is a genus of plant pathogens responsible for severe damage to crops, natural vegetation and forestry worldwide. Accurate detection, diagnosis and species identification is fundamental to disease management, clear scientific communication and the statutory measures to prevent pathogen spread and yet this often proves challenging. Many serious disease problems have
emerged in recent years, often associated with previously undescribed Phytophthora species and damaging plants in both commercial production systems and natural ecosystems. Many advances in DNA-based molecular diagnostics and DNA sequencing have been made recently that increased our ability to accurately detect and characterise Phytophthoras. However, there is a
continual need for improvement and an increasing interest in a broader monitoring of Phytophthora species in natural ecosystems to get a better feel for their distribution and impact through understanding their ecology.
This review examines the technical advances in the field and the rationale for such studies on Phytophthora.
Abstract: In Western European cropping rotations, volunteer potato plants are a major weed problem, mainly because they spread Phytophthora infestans, which causes late blight in potato crops. The manual labour that is required for control of these plants can be reduced by using automated detection and control of volunteer potato plants in sugar beet fields. Machine vision was used to detect volunteer potato plants in real time conditions at a travel speed of 0.8 m s-1. Dose effect studies were performed to determine the right dose for control of volunteer plants with micro-sprayer-applied glyphosate in a gel. In a field trial the biological efficacy of the system was at the level where it controlled 84% of the volunteer potato plants.
Abstract: Studies were performed to investigate the changes of potato pathogenesis-related proteins (PRPs) upon infection with late blight pathogen. Obtained data showed that in both resistant and susceptible potato cultivars, inoculated leaves with Phytophthora infestans showed a significantly higher amount of total protein than the healthy ones. In a bioassay experiment, the crude protein extracted from leaves of resistant potato cultivars showed the lowest, while those from susceptible ones indicated the highest fungal growth. SDS-PAGE analysis of acidic soluble proteins extracted from inoculated potato leaves at different periods of inoculation with P. infestans showed that nine proteins with molecular weight ranged from 12-45 kDa were increased gradually with time. RT-PCR analysis showed that in spite of the variation between tested potato cultivars in their resistance to late blight disease, the gene encoding osmotin-like protein (OSM-1 gene) existed at DNA level. At RNA level, an induction of OSM-1 gene expression was detected potato cultivars after inoculation with P. infestans. The expression of OSM-1 gene in the resistant cultivar occurred earlier (6 hpi) and stronger, while being induced later in the susceptible cultivar. Earlier and higher accumulation of PRPs in the resistant cultivars suggested that they were related to the defense mechanism against P. infestans.
Abstract: In the present work, we investigated the specificity and sensitivity of serological and molecular tools for the detection of Phytophthora infestans in infected tissues of susceptible and resistant potato cultivars and to study disease development among these cultivars. Serological tools [enzyme-linked immunosorbent assay (ELISA) and dot blot immunoassay (DIA)] were performed using antiserum raised from soluble mycelial protein of P. infestans, while molecular tool [Polymerase chain reaction (PCR)] was performed using specific primer to P. infestans, which designed on the basis of internally transcribed spacer1 ITS1 (GenBank accession no AY770739). Cross-reactivity of the antiserum was tested against 50 µg antigens of 9 isolates of P. infestans and antigens of Fusarium sp., Pythium sp., R. solani, M. phaseolina, A. solani, E. carotovora and R. solanacearum. Results showed that the antiserum was adequate specificity among different antigens of the tested isolates of P. infestans. On the other hand, PCR amplification indicated that, all the P. infestans isolates amplified a product of approximately 813 bp with the primer. While no amplification products were obtained with the other tested genera. The detection sensitivity of the antiserum ranged from 5-50 µg of P. infestans antigen. The PCR assay was also very sensitive for detecting P. infestans, only 1 pg of purified DNA or DNA isolated from 500 sporangia was needed to detect the pathogen. Applications of serological and molecular tools were effective to detect the pathogen in infected symptomatic, asymptomatic potato tissues (Leaves & tubers) and can also provide important information on P. infestans-potato interaction and disease development.
Abstract