نوع مقاله : مقاله پژوهشی
نویسنده
بخش تحقیقات گیاه‌پزشکی، مرکز تحقیقات و آموزش کشاورزی و منابع طبیعی خراسان رضوی، سازمان تحقیقات، آموزش و ترویج کشاورزی، مشهد، ایران
چکیده
کلیدواژه‌ها
موضوعات
عنوان مقاله [English]
نویسنده [English]
Introduction
Esca of grapevine (Vitis vinifera) is an important complex disease in almost all areas, where grapevines are grown. The symptoms of this disease may affect the trunk, branches, shoots (brown wood-streaking and white rot of trunk), leaves (light green or chlorotic, irregular areas between the veins or along the leaf margin, which gradually spread from the basal to the distal parts of the shoot) and fruit (tiny brown spots and sometimes wilt of berries). Toxins are secondary metabolites, which are important virulence factors of phytopathogenic fungi. Phaeoacremonium aleophilum (Pal), Phaeomoniella chlamydospora (Pch), and Fomitiporia mediterranea (Fme) produced two pentaketides (scytalone and isosclerone), and the α-glucan named pullulan. Several evidences indicated that at least some of these metabolites may induce the characteristic symptoms of diseases. The main objective of this research was to establish a clear efficient and cheap method using callus and extracted toxic metabolites in order to select susceptible, tolerant, or resistant commercial grapevine cultivars to the esca disease.
Materials and Methods
Esca-associated fungi were obtained from the Agricultural Research, Education and Extension Organization (AREEO), Khorasan Razavi province, Iran. Five mL of a suspension of three mentioned fungi (10-day-old) in 50 ml sterile water were added to 1 L flask containing 150 mL Czapek Dox medium amended with 0.1% yeast and 0.1% malt extract. They were incubated at 25°C for 28 days in the dark. The mycelia were removed by filtration using Filter membranes, nitrocellulose 0.22 μm. The toxic secondary metabolites were extracted from this suspension. Briefly, the culture filtrates from each fungus (2 L per strain) were treated with equal volumes of cold ethanol-acetone and incubated in an ice batch for 4 to 6 hours. The resulting precipitates were extracted by centrifugation at 3000 rpm for 10 seconds. Recrystallization was done by dissolving the polymer in hot water and adding the same volume of ethanol to it, and the formed precipitate was filtered through a Whatman filter, dried at 40°C and weighed. The toxic metabolite obtained from liquid cultures of each fungal species was assayed on detached leaves of 5 grapevine commercial cultivars of Khorasan Razavi province, including Torkaman8(TU8), Kolahdari(KOL), Torkaman6(TU6), Fakhri Shahrood(FSH), and Keshmeshi-Quchan(KQU). The leaves with their petioles were immersed in a 3 mL solution until complete absorption, which usually took a few hours, and then were transferred to distilled water. Callus of the five grapevine cultivars, micro propagated shoot cultures were cultivated on modified MS media containing 15, 30, and 45% toxic metabolites. The grown callus was inoculated with different concentrations of toxic metabolites, then dry weight of callus and vital cells were measured visually, as well as spectrophotometrically, by using Triphenyl Tetrazolium Chloride (TTC).
Results and Discussion
The Pal, Pch, and Fme fungi were all able to produce toxic secondary metabolites, but an isolate of the Pal fungus obtained from Memlejeh (a village in the Central District of Bojnord County, North Khorasan Province, Iran.) produced a large amount of pullulan. The symptoms produced on the detached leaves that absorbed a toxic solution were quite similar to those observed on the leaves of the same cultivars naturally infected by the same fungal species. Differences between cultivars in symptom severity were also observed under experimental conditions. The results showed that reduction of the callus dry weight in TU8 grapevine cultivar was the lowest (26%) compared to the other cultivars tested, followed by KOL (31%) and TU6 (40%) cultivars. The KQU and FSH cultivars showed the highest reduction in callus dry weight (48% and 44%, respectively). The effect of pullulan produced by three important esca-associated fungi indicated that the toxin produced by the Fme fungus had the least effect and the Pal fungus showed the greatest effect in reducing dry weight of the callus. Dry weight of the callus in 15% concentration of the toxic metabolite showed the least decrease and maximum decrease of the callus weight was obtained in 45% concentration. Among the cultivars tested, the callus cells of Torkaman 8 had the highest optical density, which means the number of living cells was more than other cultivars. The KQU cultivar with minimal live callus cells showed more sensitivity to the metabolite. Toxin of the Fme fungus had the least and that of the Pal fungus had the most effect on the callus cells. The percentage of survival rate in the inoculated callus cells of Torkaman 8 compared to the KQU cultivar at 45% concentration of the extract was about 50.36% and the survival rate in the KQU cultivar was about 15.51%. Also, the obtained results showed that the grapevine cultivar TU8 was comparatively more tolerant than other cultivars tested. On the other hand, the FSH and KQU cultivars were susceptible to secondary metabolite of the mentioned fungi.
Conclusions
Occurrence of the esca diseases is increasing in grapevine all over the world, whereas efficient therapeutic strategies are lacking. The use of toxic metabolites of esca-associated fungi and tissue culture of the host plants under in vitro conditions could be cheap,easy, and helpful assays for controlling the disease via selection, improvement, and reproduction of the tolerant genotypes. According to the reaction of grapevine cultivars to the secondary metaboilte of esca-associated fungi, it can be concluded that the TU8 grapevine cultivar is less sensitive than the KQU grapevine cultivar against the esca disease.
کلیدواژه‌ها [English]
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©2024 The author(s). This is an open access article distributed under Creative Commons Attribution 4.0 International License (CC BY 4.0). |
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