Amer Dababat

Development of two species-specific primer sets to detect the cereal cyst nematodes Heterodera avenae and Heterodera filipjevi

Twelve Heterodera species are of major economic significance in wheat and barley. Of these, H. avenae, H. filipjevi and H. latipons are among the most important ones, and sometimes coexist. The identification of Heterodera species using morphological characteristics is time consuming, requires specialized skill and can be imprecise, especially when they occur mixed in field populations. Molecular techniques can provide a more accurate way for nematode identification. This study reports the results of experiments targeting the mitochondrial cytochrome oxidase subunit 1 (COI) gene to develop species-specific primers that could be used for the identification of H. avenae and H. filipjevi. The COI gene of 9 Heterodera spp. and Punctodera punctata was partially sequenced and the resultant sequences were aligned to find unique sites suitable for the design of primers. The alignment showed variability between H. avenae, H. filipjevi and other Heterodera species. Two sets of species-specific primers were identified for the identification of both species and the conditions for their use in PCR were optimised. The specificity of the designed primers was checked by comparison with one population of P. punctata and populations of 14 other Heterodera species, nine populations of H. avenae and 10 populations of H. filipjevi originating from different countries. To test the sensitivity, the PCR was run with DNA extracted from five second-stage juveniles (J2) of H. avenae or five J2 of H. filipjevi mixed with DNA extracted from varying numbers of J2 of H. latipons. It was possible to detect as few as five J2 of H. avenae or H. filipjevi among 100 J2 of H. latipons. The two primers sets allow the detection of H. avenae and H. filipjevi where they occur in mixed populations with other Heterodera spp.

Development of a species-specific PCR to detect the cereal cyst nematode, Heterodera latipons

Summary ‐ Several Heterodera species can reduce the yield of wheat and barley, among which H. avenae, H. filipjevi and H. latipons are economically the most important. Their identification, based on morphological characteristics, is not straightforward but can be made easier using molecular techniques. In this study, we developed species-specific primers for the detection of H. latipons. The actin gene of eight Heterodera species was partially sequenced and, after purifying and sequencing the PCR products, all sequences were aligned to find unique sites. The alignment showed moderate to very high similarities between the species. However, a small fragment of the actin gene was suitable for the construction of a potentially useful species-specific primer for H. latipons. The optimised PCR was subsequently tested with several populations of 14 Heterodera species and a single population of Punctodera punctata. Heterodera latipons was represented by 16 populations originating from six different countries. The primer set (Hlat-act), designed using AlleleID 7.73, was shown to be very specific. To test its sensitivity further, the PCR was conducted on DNA extracted from five second-stage juveniles (J2) of H. latipons mixed with five or 100 J2 belonging to H. avenae. The PCR was able to detect up to 1:10 dilution of the DNA obtained from five J2. The results showed that a specific and sensitive H. latipons species-specific PCR was constructed.

Identity and pathogenicity of Fusarium species associated with crown rot on wheat (Triticum spp.) in Turkey

An extensive survey was carried out to collect Fusarium species colonizing the lower stems (crowns) of bread wheat (Triticum aestivum L.) and durum wheat (T. durum Desf.) from different wheat growing regions of Turkey in summer 2013. Samples were collected from 200 fields representing the major wheat cultivation areas in Turkey, and fungi were isolated from symptomatic crowns. The isolates were identified to species level by sequencing the translation elongation factor 1-alpha (TEF1-α) gene region using primers ef1 and ef2. A total of 339 isolates representing 17 Fusarium species were isolated. The isolates were identified as F. culmorum, F. pseudograminearum, F. graminearum, F. equiseti, F. acuminatum, F. brachygibbosum, F. hostae, F. redolens, F. avenaceum, F. oxysporum, F. torulosum, F. proliferatum, F. flocciferum, F. solani, F. incarnatum, F. tricinctum and F. reticulatum. Fusarium equiseti was the most commonly isolated species, accounting for 36% of the total Fusarium species isolated. Among the damaging species, F. culmorum was the predominant species being isolated from 13.6% of sites surveyed while F. pseudograminearum and F. graminearum were isolated only from 1% and 0.5% of surveyed sites, respectively. Six out of the 17 Fusarium species tested for pathogenicity caused crown rot with different levels of severity. Fusarium culmorum, F. pseudograminearum and F. graminearum caused severe crown rot disease on durum wheat. Fusarium avenaceum and F. hostae were weakly to moderately virulent. Fusarium redolens was weakly virulent. However, F. oxysporum, F. equiseti, F. solani, F. incarnatum, F. reticulatum, F. flocciferum, F. tricinctum, F. brachygibbosum, F. torulosum, F. acuminatum and F. proliferatum were non-pathogenic. The result of this study reveal the existence of a wide range of Fusarium species associated with crown rot of wheat in Turkey.