Commercial cultivars, including ‘Longshu 3’, ‘Longshu 5’, ‘Longshu 6’, ‘Longshu 7’, ‘Shuixihong’, ‘Sepihong’, ‘Zihuabai’, ‘LK99’, ‘Kexin 1’, ‘Heimeiren’, ‘Qingshu 168’ were bred in China. ‘Desiree’ and ‘Favorita’ bred by Hollander ,‘Atlantic’ and ‘Shepody’ breeded by USA and Canada, and potato lines J08-1, J08-2, J08-4, J07-2, and J07-5 which were provided by obtained from the Japanese Hokkaido University. These materials were all virus free assured by using tissue culture transfer, and maintained in the Research Center of Potato Breeding in Inner Mongolia Agricultural University, Hohhot, China.
Rhizoctonia solani AG2-1 strain WC-16 was isolated from an infected potato tuber from a field in Wuchuan county of Inner Mongolia where potato, which was confirmed to be highly pathogenic on stems and tubers of ‘Atlantic’ potato. The fungal culture was stored on potato sucrose agar (PSA) at 4℃ for later use. To prepare the inoculum, wheat bran (100 g) was soaked in distilled 200 ml water in a 500 ml conical flask, and autoclaved at 121℃ for 40 min. Each flask was inoculated with 10 discs (0.8 cm diameter) of 4-day-old R. solani culture, and incubated at 25℃ in the dark for 30 days. The wheat bran mixed with mycelia and a pseudosclerotia of R. solani was air dried and then manually rubbed into fine pieces.
Inoculum-based evaluation on potato resistance to R. solani under field conditions
Field trials were conducted at two locations. In 2009, the trial was conducted in the Horticulture Science and Technology Demonstration Area of Hohhot City. potato seed tubers of different potato germplasms were planted in the field covered with a nylon screen in May. Soil properties were analyzed by the Testing Center of Agricultural Products Quality and Safety, Hohhot, Inner Mongolia. The field is composed of light sandy soil with 18.5 g/kg organic matter, 74 mg/kg hydrolysis nitrogen, 23.0 mg/kg available phosphorus, and 116 mg/kg rapidly available potassium. Prior to planting, potato tubers were disinfested for 20 min with 0.5% KMnO4, followed by rinsing with tap water and cutting into 30 to 50 g seed-tuber pieces. At the seeding site, 0 (control), 2, 3, 4, or 5 g of wheat bran inoculum was applied per seed piece, followed by planting. All 20 potato germplasms were planted in a randomized complete block design, with two rows per germplasm, and 20 plants per row. Plant space was 30 cm within rows and 60 cm between rows. Each treatment had three replications. During the growth period of 133 days, the potato entries were not fertilized but irrigated three times.
In May of 2010, the trial was repeated at the Inner Mongolia Agricultural University Farm, where there was higher soil fertility than that in 2009. Soil properties were as follows: 18.7 g/kg organic matter, 75 mg/kg hydrolysis nitrogen, 24.50 mg/ kg available phosphorus, and 118 mg/kg rapidly available potassium. The wheat bran inocula were applied at 0, 2 and 4 g per seed piece.
Sixty-five days after planting, potato underground stems were dug out and examined for canker. Total of 100 stems were processed. Disease severity was expressed as following rating scale based on the percentage of lesion on the stem : 0 (no lesion), 1 (1–5%), 2 (6–25%), 3 (26–50%), 4 (51–75%), and 5 (76–100%). Disease index (DI) was calculated as: DI = 100 × Σ(ri x ni)/(N × 5), where N = total number of plants evaluated, r is the level of severity from 0 to 5, and i = specific level of severity (from 0 to 5), n = number of corresponding grade plants evaluated. Relative resistance index (RRI) was calculated as: RRI = 1- DIX/DImax, where DIx = disease index of the observed stem and DImax = the maximum disease index of all cultivars or lines in the same repetition. Stem canker resistance was measured with the relative resistance index (RRI) as follows: 1: immune (I); 0.99 to 0.80: highly resistant (HR); 0.79 to 0.50: moderately resistant (MR); 0.49 to 0.20: moderately susceptible (MS); and 0.19 to 0.00: highly susceptible (HS).
Potato resistance evaluation using RS toxin
RS toxin derived by heating. Fifty milliliters of improved Richard medium [28–29] was added into a 250 ml conical flask, and autoclaved for 20 min. After cooling down, each flask was inoculated with 5 discs (0.5 cm diameter) of 4-day-old R. solani culture, and incubated for 20 days at 25℃ in the dark, with hand shaking daily. After incubation, the culture was filtered through a filter paper at 50 µm pores. The filtrate was examined for absorbance peak at wavelength 258.8 nm to confirm the presence of toxin, and collected for later use. The filtrate was added into agar at concentration 0.4% by volume, and poured into test tubes (18 cm × 1.8 cm) at 12 ml/tube, and autoclaved for 20 min at 115℃. This was used as toxin derived by heating . The trial was repeated once.
RS toxin derived by active carbon adsorbtion. Equal amount of liquid active carbon was added into the filtrate as described above, and kept for 12 h at 4℃, followed by centrifugation for 15 min at 3500 r/min. The precipitated pellet of active carbon was mixed with equal amount of methanol, and then concentrated with a rotary evaporator at 45℃, which was yellow slurry. It was diluted with distilled water to the original volume, then filtrated through 0.22 µm membrane for sterilization. The derived product was used a toxin by adsorption . The absorbance peak of toxin was examined at wavelength 258.8 nm for confirmation of toxin and poured into sterilized test tubes (18 cm × 1.8 cm) at 12 ml/tube contained agar at concentration of 0.4%. The trial was repeated once.
Disease evaluation. Virus-free potato ‘Atlantic’ seedlings were grown in Murashige and Skoog medium until their height reached around 12 cm. The seedlings were cut into stem sections with one leaf bud attached per stem section. This was a standard for stem section used for inoculation throughout the study, unless otherwise stated. The stem sections were placed into media amended with one of the toxins prepared either by heating or absorption. MS medium without toxin was used for control. Total of 60 stem sections were used for each treatment, which was replicated three times. After incubation at 25℃ with 16 h light (4000 lux)/day arrangement, the seedlings were observed for necrotic symptom from four to eight days of incubation, seedlings length were measured and growth inhibition rate was calculated after eight days. Growth inhibition = (seedling length of control - seedling length of treatment) / seedling length of control × 100%.
Comparison of inoculation with Rhizoctonia solani inoculum and its toxin. Water agar (0.4%) was autoclaved and cooled to 40℃, mycelia of R. solani in above culture were collected and ground into small pieces in a mortar with a pesto using aseptic operation, then added into the water agar with equal volumes culture, and shaken well. This suspension was used as a pathogen inoculum. The pathogen inoculum and toxin derived by heating containing 0.4% agar were separately put into test tubes (18 cm × 1.8 cm) at 12 ml/tube. The seedling of 12-cm long and stem sections of potato ‘Atlantic’ were transferred into tubes containing pathogen inoculum, toxin, and MS medium. There were 60 plants per treatment. The tubes were incubated at 25℃ with 16 h light (4000 lux)/day. The seedlings and stem sections were observed for necrotic symptom from four to eight days of incubation. Seedlings and stem section length were measured and growth inhibition was calculated. The trial was conducted three times.
Effects of toxin concentration on symptom expression. The above filtrate was diluted into 1/2 and 1/4 concentrations with either MS liquid medium or distilled water. To concentrate the toxin, the filtrate was treated with heat to obtain 2 or 4 times of concentration. The prepared different concentration toxin were added into 0.4% agar in tubes at 12 ml/tube, then autoclaved at 115℃ for 20 min. MS medium and distilled water agar were used as a control. Seedlings and stem sections of ‘Atlantic’ were transferred into the tubes containing different concentrations of toxin. In another trial, the filtrate was made into toxin and toxin of 3/4 concentration with distilled water. Distilled water agar was control. Potato seedlings and stem sections of 12 cultivars were transferred into different concentration toxin.
There were 60 plants each treatment. Then incubated at 25℃ with 16 h light (4000 lux)/day, the symptoms were observed for necrotic from four to eight days of incubation, seedlings length were measured and growth inhibition rate was calculated after eight days. The trials were conducted three times.
Resistance Identification Of Potato Cultivars Using Toxin-based Assays
After preliminary trials, optimized assay was determined as follow. Virus-free potato seedlings were grown in Murashige and Skoog medium until their height reached around 12 cm. The seedlings were transferred into a medium amended with heat-derived toxin, with one seedling per tube. Distilled water agar was used as a control. Total of 60 seedlings were transferred for each variety or line, which was replicated three times. After incubation at 25℃ with 16 h light (4000 lux)/day, the seedlings were observed for necrotic symptom from four to eight days of incubation, and disease was measured after eight days. The disease was scored using the following scale based on the percentage of girdled stem and wilted or dead leaves: 0 (no girdled stem and wilted or dead leaves), 1 (1–5%), 2 (6–25%), 3 (26–50%), 4 (51–75%), and 5 (76–100%). Disease index (DI), relative resistance index (RRI), and seedling resistance measured using relative resistance index (RRI) were as described above in the field trials.
Data were analyzed using the SPSS 17.0 statistical software (IBM SPSS Statistics for Windows, Version 22.0, IBM Corp. Armonk, NY, USA). GLM procedure was used for analysis of variance. LSR multiple comparisons was used for mean separation, at significance level 0.05.