Antoaneta B. M- Kroumova; Ivan Artiouchine; George Wagner. KTRDC, Lexington, KY, USA

Transcription

1 Antoaneta B. M- Kroumova; Ivan Artiouchine; George Wagner KTRDC, Lexington, KY, USA

2 Introduction Black shank is the largest annual disease threat to Kentucky tobacco, surpassing blue mold in importance, if we look at annual revenue losses. The disease is caused by the soil fungus Phytophthora parasitica, var. nicotianae that attacks plant s roots and stems and leads to dark-brown to black lesions, eventually killing the plant. Currently, P. parasitica is found in the majority of tobacco fields in the southern United States, causing damage in 2 to 5.5% of them, but in some fields losses can be 1%. Kroumova_CORESTA_15 2

3 P. parasitica life cycle Gallup, C.A., M.J. Sullivan, and H.D. Shew. 6. Black Shank of Tobacco: The Plant Health Instructor. DOI: 1.194/PHI-I Infection occurs through the roots and is favored by moisture and warm temperatures Kroumova_CORESTA_15 3 The are two races of this fungus on the territory of Kentucky: race and race 1

4 Approaches to fight the disease: Breeding of resistant varieties (e.g. burley lines 9 LC and 6 LC, MS KY14xL8 or MS Burley 21xL8) L8 hybrids are resistant only to Race. Resistance is conditional. Fungicide application (mefenoxam-containing fungicides) Long-term negative effect: acquired resistance to them, collateral toxicity and environmental pollution. Crop rotation Biological approach - still experimental (Rhizoctonia fung; Paenibacillus polymyxa). Kroumova_CORESTA_15 4

5 Continue Because of the long-term negative effect of the fungicides, there is a need for development of alternative ones that are sustainable and eco-friendly. Some plant-made products are shown to have antibacterial, antifungal and insecticidal properties. Some essential oils and tobacco root constituents have been found effective against black shank disease. Kroumova_CORESTA_15 5

6 Hypothesis: Our laboratory is specialized in the study of surface chemistry of tobacco leaves. Recently, it was discovered that a tobacco trichome-produces glycoprotein (T- phylloplanin) is active against blue mold spores. T-phylloplanin and trichome-produced diterpene cis-abienol showed a potential for inhibit black shank in laboratory tests. We hypothesized that application of these natural products, and also of terpenoids sclareol and labdenediol, in potted plants in the green house can inhibit P. parasitica zoospore penetration into the roots and can prevent/reduce disease symptoms. Kroumova_CORESTA_15 6

7 Sites of synthesis of cis-abienol, T- phylloplanin and labdenediol N. tabacum T.I.168 is known to produce both - T-phylloplanin and also terpenoids, such as cis-abienol or labdenediol in quantity. Gum producing, including cisabienol and labdenediol T-phylloplanin producing SDS-PAGE in 15% acrylamide Phylloplanin bands Kroumova_CORESTA_15 7

8 Natural products collection T-Phylloplanin: T.I. 168 leaf water wash, followed by concentration via freeze-drying. cis-abienol: We used a line of T.I. 168, created in our lab, that produces increased (at least 3 times) amount of cis-abienol. The leaf exudate was acetonitrile washed, concentrated, and further purified by partitioning between hexane and % methanol. Labdenediol: We used a line of T.I. 168, created in our lab, producing labdenediol in high abundance (9% of all terpenoids). The compound was collected the same way as cis-abienol, but was not purified additionally. Sclareol: Obtained commercially. Kroumova_CORESTA_15 8

9 cis-abienol purification std 14% cis-abienol LD SE A A. Crude acetonitrile wash 7.16 min cis-abienol min Sugar esters 1.34 min Labdenediol 4.67 min - standard Std. 85% cis-abienol B B. Purified cis-abienol and structure 7.14 min cis-abienol 4.65 min - standard Kroumova_CORESTA_15 9

10 A customized method for P. parasitica spores preparation One plug (1 3 cm) of agar-mycelium from a 3-7 d old culture is placed in a plate with V8 medium. The plate is kept in the dark for 4-6 days. 5 plugs (1 3 cm) of agar-mycelium from the edge of the mycelial mat are placed in a Petri dish with ml sterile tap water. The plate is kept in the dark, at room temperature for 7 days. Hyphal mat is monitored under scope for sporangia formation. Upon formation, sporangia are induced to release spores by temperature stress: 4 o C for 3 min, 37 o C for 8 min, and 22 o for min. Moving spores are monitored under microscope. Spores are immobilized and counted with hemocytometer. Kroumova_CORESTA_15 1

11 Establishing the working concentrations of spores 1 KY14 KY14 x L8LC 9 LC Spore concentration (x1 3 )per ml P. parasitica race 1 spores were diluted with water to the desired concentration and 1 ml of each concentration was applied to the base of the stems of 5 weekold seedlings. Kroumova_CORESTA_15 11

12 Establishing the working concentrations of natural products Phylloplanin cis-abienol mg powder/ml 6 mg/ml Sclareol 1 3 Kroumova_CORESTA_15 12 mg/ml

13 Effect of sclareol, phylloplanin and cis-abienol on suppression of black shank disease in 7 week-old KY14 plants 1 Race Race 1 Water control Spores Sclareol +spores Treatment Phyllo +spores cis-abienol +spores Kroumova_CORESTA_15 13

14 The natural products delayed/suppressed black shank symptoms in 7 week-old KY14 race spores race 1 spores 1 water contr* spores Scl+spores* Phyllo+spores cis-abienol 1 water contr* spores Scl+spores Phyllo+spores cis-ab+spores Days post infection Days post infection Kroumova_CORESTA_15 14

15 Observation of the disease symptoms in KY14 seedlings spores phyllo 1** MeOH sclareol cis-abienol Race spores phyllo sclareol MeOH *Pictures were taken on the 1 th day post-infection **Numbers represent mg/ml cis-abienol Race 1 Kroumova_CORESTA_15 15

16 Effect of sclareol, phylloplanin and cis-abienol on suppression of race 1-caused disease in 7 week-old KY14xL8* Race Race 1 controlwater spores Sclareol +spores Phyllo +spores cis-abienol +spores Treatment 1 Disease development Days post-infection Kroumova_CORESTA_15 water contr* spores Scl+spores* Phyllo+spores cis-abienol 16

17 Effect of sclareol, phylloplanin and labdenediol on suppression of black shank disease in 8 week-old KY controlwater spores Labdenediol +spores Treatment Sclareol +spores Phyllo +spores Race Race 1 Kroumova_CORESTA_15 17

18 Delay of black shank symptoms in 8 weekold KY14 A 1 race spores B 1 race 1 spores Days post-infection controlwater spores LD Scl Phyllo Days post-infection controlwater spores Kroumova_CORESTA_15 18 LD Scl Phyllo

19 Delay of race -caused disease by labdenediol and phylloplanin, and disease prevention by sclareol spores LD 3* Scl Scl Phyllo 1 *mg/ml spores LD Scl Scl Phyllo 13th day PI Kroumova_CORESTA_ th day PI

20 Conclusions: Sclareol appeared to be the best candidate for future study on a large scale. Sclareol strongly suppressed the disease from both races in younger plants from both lines. It is commercially produced from clary sage (Salvia sclarea) at a price of ~$7-1/kg. One kg could be used to treat 25, plants. This work is the first to show that sclareol suppresses black shank disease in tobacco. cis-abienol, despite its good antifungal activity, is not feasible for large scale use due to the production and stability limitations. Kroumova_CORESTA_15

21 Conclusions (continued): Labdenediol worked well in suppressing the disease in older KY14 plants. Further studies are needed, however, with wellpurified substance. Phylloplanin is not as efficient black shank suppressor as the other natural products. Kroumova_CORESTA_15 21

22 Acknowledgments: Council of Burley Tobacco, Kentucky, USA KTRDC and BAT University of Kentucky, Lexington, KY: Dr. Natalia Martinez (KTRDC) Mr. Ed Dixon (Plant Pathology) Dr. Kenny Seebold (Plant Pathology) Dr. Victor Korenkov (KTRDC) Kroumova_CORESTA_15 22

23 Kroumova_CORESTA_15 23