Characteristics of the Infection of Tilletia laevis Kühn (syn. Tilletia foetida (Wallr.) Liro.) in Compatible Wheat

<i>Tilletia laevis</i> Kühn (syn. <i>Tilletia foetida</i> (Wallr.) Liro.) causes wheat common bunt, which is one of the most devastating plant diseases in the world. Common bunt can result in a reduction of 80% or even a total loss of wheat production. In this study, the characteristics of <i>T. laevis</i> infection in compatible wheat plants were defined based on the combination of scanning electron microscopy, transmission electron microscopy and laser scanning confocal microscopy. We found <i>T. laevis</i> could lead to the abnormal growth of wheat tissues and cells, such as leakage of chloroplasts, deformities, disordered arrangements of mesophyll cells and also thickening of the cell wall of mesophyll cells in leaf tissue. What’s more, <i>T. laevis</i> teliospores were found in the roots, stems, flag leaves, and glumes of infected wheat plants instead of just in the ovaries, as previously reported. The abnormal characteristics caused by <i>T. laevis</i> may be used for early detection of this pathogen instead of molecular markers in addition to providing theoretical insights into <i>T. laevis</i> and wheat interactions for breeding of common bunt resistance.

تقييم الفعالية البيولوجية للبكتريا المعزولة من داخل النبات في مقاومة مرضي التفحم المغطى وتعفن الجذور وموت البادرات في الحنطة Triticum aestivum L.

أجريت الدراسة لتقييم فعالية بكتريا داخل النبات في السيطرة على المسببات المرضية Pythium aphanidermatum المسبب لمرض تعفن الجذور وموت البادرات و T. caries /Tilletia laevis المسبب لمرض التفحم المغطى، إذ عزلت 50 عزلة من البكتريا من أنسجة نبات الحنطة من بعض مناطق محافظة صلاح الدين. أشارت نتائج إختبار التضاد المباشر للبكتريا المعزولة مع المسبب المرضي P. aphanidermatum إلى أن مدى التثبيط قد تراوح بين (0.3 - 9.3 ملم) إذ بلغ أعلى معدل تثبيط في العزلة SHSt2 بمنطقة تثبيط بلغت 9.3 ملم وشخصت هذه العزلة الى مستوى النوع Pseudomonas flvescens (SHSt2) . أن المعاملة (بذور + رش 1012 خلية بكتيرية/ مل) في التجربة الحقلية قد أبدت تفوقاً معنوياً في مؤشرات النبات الخضرية كمحتوى الكلوروفيل وارتفاع النباتات. أثرت المعاملة ببكتريا P.flvescensفي نسبة الاصابة بالفطرين الممرضين P.aphanidermatum و T. laevis/T. caries إذ بلغت ادنى نسبة اصابة في معاملة البكتريا (بذور + رش 1012 خلية بكتيرية/ مل) بنسبة إصابة بلغت 19.94 و11.13% على التوالي مقارنة باعلى نسبة اصابة في معاملتي السيطرة بوجود الممرضين فقط إذ بلغت نسبة الإصابة 81.57 و 58.47 % على التوالي. في المؤشرات الانتاجية ، رفعت المعاملة بالبكتريا P. flvescens من الوزن الكلي للنبات بوجود الممرضين P. aphanidermatum و T. laevis/T. caries إذ بلغ أعلى وزن 60.4 و 58.71 غم على التوالي في معاملة (بذور + رش 1012 خلية بكتيرية/ مل)، مقارنة بأدنى وزن في معاملتي السيطرة للممرضين إذ بلغ 26.47 و 20.56 غم على التوالي، إضافة إلى ذلك فقد إنعكس تأثير المعاملة بالبكتريا P. flvescens على دليل الحصاد إذ بلغت أعلى نسبة 36.5 و28.75% بوجود الممرضين P. aphanidermatum و T. laevis/T. caries على التوالي في معاملة (بذور + رش 1012 خلية بكتيرية/ مل) مقارنة بأدنى نسبة في معاملة السيطرة للممرضين إذ بلغت 22.42 و18.42 %على التوالي .

Development of droplet digital PCR for the detection of Tilletia laevis, which causes common bunt of wheat, based on the SCAR marker derived from ISSR and real-time PCR

Common bunt of wheat caused by Tilletia laevis and/or T. caries (syn. T. tritici), is a major disease in wheat-growing regions worldwide that could lead to 80% or even total loss of production. Even though T. laevis can be distinguished from T. caries on the bases of morphology of teliospores using microscopy technique. However, molecular methods could serve as an additional method to quantify the pathogen. To develop a rapid diagnostic and quantify method, we employed the ISSR molecular marker for T. laevis in this study. The primer ISSR857 generated a polymorphic pattern displaying a 1385 bp T. laevis-specific DNA fragment. A pair of specific primers (L57F/L57R) was designed to amplify a sequence-characterized amplified region (SCAR) (763 bp) for the PCR detection assay. The primers amplified the DNA fragment in the tested isolates of T. laevis but failed in the related species, including T. caries. The detection limit of the primer set (L57F/L57R) was 5 ng/µl of DNA extracted from T. laevis teliospores. A SYBR Green I real-time PCR method for detecting T. laevis with a 100 fg/µl detection limit and droplet digital PCR with a high sensitivity (30 fg/µl detection limit) were developed; this technique showed the most sensitive detection compared to the SCAR marker and SYBR Green I real-time PCR. Additionally, this is the first study related the detection of T. laevis with the droplet digital PCR method.

Development of ISSR-derived SCAR Marker and SYBR Green I Real-time PCR Method for Detection of Teliospores of Tilletia laevis Kühn

Common bunt, caused by Tilletia laevis Kühn [syn. T. foetida (Wallr) Liro] and Tilletia tritici (Bjerk.) Wint. [syn. T. caries (DC) Tul.], is an important wheat disease worldwide. To quickly differentiate the closely related fungi T. laevis, T. tritici and Tilletia controversa (a pathogen that causes dwarf bunt of wheat and has been requested as a quarantined pathogen in many countries), a rapid diagnostic and detection method for an ISSR molecular marker was developed for the first time in this study. Based on the T. laevis-specific band (1300 bp) amplified by the primer ISSR860, a pair of SCAR primers (L60F/L60R) was designed to amplify a specific 660-bp DNA fragment from the isolates of T. laevis but not other related pathogens. The detection limit of the SCAR marker was 0.4 ng/μl of DNA from T. laevis; moreover, a SYBR Green I real-time PCR method was also successfully developed based on the SCAR marker with the detection limit of 10 fg/μl T. laevis DNA. This is the first report of a rapid, specific and highly sensitive SCAR marker and SYBR Green I real-time PCR method for detection of the teliospores of T. laevis based on ISSR technology. This method allows highly efficient, rapid and accurate differentiation of the pathogen from related pathogens, especially from the very similar pathogens T. tritici and T. controversa.

Effect of inoculum doses on common bunt infection on wheat caused by Tilletia tritici and T. laevis

: In the trial with different doses of common bunt teliospores used for inoculation of wheat the number of bunted ears increased with the increasing dose of inoculum. This increase was small (statistically insignificant) in the resistant cv. Bill and high (statistically significant) in the susceptible cv. Samanta. The effect of inoculation on the vigour of inoculated plants was estimated according to the number of tillers per plot in summer in cultivars inoculated with different inoculum doses sown at the same sowing rate in autumn. The number of tillers was decreasing with the increasing inoculum dose. This was less pronounced in the resistant cv. Bill than in the susceptible cultivar Samanta. Competition between Tilletia tritici and Tilletia laevis during infection was studied by inoculation with a 1:1 mixture of teliospores of these species and scoring bunted ears. Ears bunted with T. laevis prevailed in almost all trials. The germination test of teliospores showed a higher speed of germination of Tilletia laevis teliospores. The number of teliospores per seed after inoculation was counted. No significant difference in the number of teliospores per seed after inoculation with T. tritici or T. laevis was ascertained.

Reaction of spring wheat cultivars to common bunt caused by Tilletia tritici (Bjerk.) Wint. and Tilletia laevis (Kühn)

In 2005, 2006 and 2007 nineteen, eight and nine spring wheat cultivars, respectively, were tested in field trials for resistance to common bunt after inoculation with bunt teliospores. Nine spring wheat cultivars were tested in a greenhouse under favourable conditions for the bunt infection. Bunt incidence in the field trials varied between 0% and 38.7%, in the greenhouse between 52.9% and 100%. The results of individual cultivars and years fluctuated. A reduction in plant height, ear length, root system and increased tillering were registered in the inoculated plants. Spots on the leaves of inoculated plants were observed in three out of the four greenhouse experiments.