Sunfl ower Inbred Lines Screening for Tolerance to White Rot on Stalk

Seventy sunfl ower (Helianthus annuus L.) inbred lines were screened for tolerance to white rot on stalk. Plants were inoculated at the budding stage, with 4-day old Sclerotinia mycelium grown on PDA medium. Mycelium was placed on the leaf top and covered with tin foil, and the leaf was put into transparent nylon bag in order to maintain high humidity. Spot length on leaf was measured and plant tolerance was determined at the full fl owering stage. Obtained results were analyzed by analysis of variance. Tested lines showed signifi cant variability in response to disease. Eleven lines had lesion length less than 50% compared to susceptible control, so they could be considered tolerant and potentially interesting for breeding program.


Introduction
Sclerotinia sclerotiorum (Lib.)De Bary is omnivorus fungi and the causal agent for diseases on more than 400 plant species (Boland & Hall 1994).The fungus had been fi rst identifi ed on sunfl ower in 1861 (Purdy 1979) while in Serbia it was fi rst described in 1948 (Aćimović 1998).
It is common and widespread on sunfl ower and can attack all parts of plant.Midstalk rot is one of several forms of disease on sunfl ower and it is incited by the airborne ascospores.These spores are produced in apotecia developed on sclerotia in surface layer of soil.Dependence of apotecial formation on high humidity in the fi rst few centimeters of soil for a certain period of time greatly limits regular appearance of disease, particularly in regions with drier climate.Disease could appear from seedling to maturity, but most commonly after period of fl owering when the most devastating symptom is logging (Gulya et al. 1997).In high S. sclerotiorum infested sunfl ower fi elds under favourable conditions number of infected plants can reach up to 100% (Maširević & Gulya 1992, Rashid 1993).

Complete resistance has not yet been observed
and partial resistance is under quantitative control (Bert et al. 2002, Mestries et al. 1998).There has been a great effort in searching for tolerance to midstalk rot both in cultivated sunfl ower and wild sunfl ower species.Hence, several methods of artifi cial inoculation have been developed (Degener et al. 1998, Castano et al. 1992b, Maširević & Gulya 1992, Grauert et al. 1980, Castano et al. 2002, Vasić et al. 2004).To this date, a number of sunfl ower lines and hybrids with various levels of tolerance have been reported (Reimonte & Castano 2008, Rönicke et al. 2004, Vear 2004), but level of tolerance is not yet considered adequate for control of the disease (Fick & Miller 1997).
The aim of this study was to determine variability in response to S. sclerotiorum infection and to fi nd tolerance within selected sunfl ower inbred lines.

Sunfl ower Inbred Lines
Seventy sunflower (Helianthus annuus L.) inbred lines were screened for tolerance to midstalk rot (Tab.1).Tested inbred lines were created at Institute of Field and Vegetable Crops, Novi Sad, Serbia and have diverse origin.There was no prior information about their response to S. sclerotiorum infection of Phytopathology / Fitopatologija www.nsseme.com/journal.html( ) stem.Inbred line NSL 21, which was found to be highly susceptible to midstalk rot in field conditions (data not shown), was used as susceptible control.

Experimental Plan
The experiment was conducted on experimental fi eld of Institute of Field and Vegetable Crops at Rimski Šančevi.The experiment was designed as completely randomized block system with three replications.Each experimental unit consisted of two 3.6 m length rows.Row spacing was 0.7 m and space between plants in the row was 0.25 m.Seeds of inbred lines were sown manually, three seeds per spot, in the fi rst tenday period of April and thinned when plants fully developed fi rst true pair of leaves.

Inoculum and Inoculation
Four-day old Sclerotinia mycelium grown on PDA medium was used for the inoculation.The fungal isolate used in this study was collected from infected stems of the plants grown at the same fi eld where the test was performed.(Mestries et al. 1998).
Mycelium plugs from the edge of colony were placed on the leaf top and covered with tin foil, and the leaf was put into transparent nylon bag in order to achieve high humidity (Degener et al. 1998).In addition to this, experimental fi eld was irrigated using sprinklers three times a week with average rate of 15 mm of water per week.

Data Recording and Statistical Analysis
Lesion length on leaf was measured periodically after inoculation and plant tolerance was determined at the full fl owering stage, while percentage of plants without symptoms was recorded.Line NSL21 was used as a susceptible control.Statistical analysis was done using software Statistica 9.0.

Results and Discussion
Inoculation procedure was successful in 57 out of 70 tested inbred lines including control line, while other 13 lines had very low infection rate (Tab.1).The lines with low infection rate were excluded from the experiment.
Average length of lesion on leaves was 6.25 cm for the most tolerant line and 29.13 cm for the most susceptible line (Fig. 2).The boxwhiskers plots for lines NSL52, (being the most tolerant one), NSL57, NSL24, NSL12, NSL16, NSL4, NSL53, and NSL50 show high value of standard error.This means that data varies in that extent that certainty for conclusion about tolerance/susceptibility reaction is greatly reduced.Furthermore, great number of outliers in case of lines NSL66, NSL67, NSL41, NSL21, NSL38, NSL49 also makes it diffi cult to reach a conclusion on reaction of the line in question (Fig. 2).These lines should be retested in order to achieve more coherent results.The existence of variability between different genotypes marked as moderately susceptible and susceptible could be seen from both fi gures.This type of reaction to S. sclerotiorum could be explained by inheritance of tolerance to this fungus.Studies have shown that tolerance is partial and polygenic, and levels of tolerance vary continually (Vear & Tourvieille 1988, Achbani et al. 1996).
Breeding sunfl ower for tolerance to S. sclerotiorum is connected to various diffi culties.The fungus causes rot on roots, stem, terminal bud and head, and all these are considered as separate diseases.Tolerance is mainly controlled by additive genes and screening for tolerance relies on various methods of artifi cial inoculation.
Suitable methods for artifi cial inoculation were developed in order to successfully test breeding material for disease tolerance in most cases.A good artifi cial inoculation test should produce high infection rate, be easy to handle and imitate natural disease incidence (Degener et al. 1998).Necessity of artifi cial inoculation of sunfl ower with S. sclerotinia is due to its high dependence on the environmental conditions for natural infection.Reliance on natural attack of fungus often results in inhomogeneous infection and could produce unreliable results (Serre et al. 2004).Natural attack is highly dependent on high humidity when disease progress is rapid and destructive, whereas dry period immediately after infection could completely stop the disease (Tourvieille et al. 1992).In our study there was complete absence of natural attack in the experimental fi eld, presumably due to high temperatures and low RH during period when sunfl ower plants are most susceptible to mid-stalk rot.This indicates possible escape mechanism when there is source of inoculum but environmental factors are limiting appearance of the disease.Contrary to this, disease development was recorded on artifi cially inoculated leaves.
For the statistical analysis of lesion length we used only data from successfully inoculated plants.Leaf lesion is a reliable trait for screening against Sclerotinia and it is relatively simple compared with tests using ascospores on capitula (Castano et al. 1992b, Becelaere & Miller 2004), and tests using mycelium on capitula in growth chamber (Vear & Guillman 1977).However, reliability of this trait was not confi rmed in the study of Degener et al. (1998) who found measurement of stem lesion more adequate.
Although different authors measured leaf lesions 4 to 7 days after inoculation (Degener et al. 1998, Castano et al. 1992a, Castano et al. 1992b, Caceres et al. 2006), in our case it was not possible due to slow progression of leaf lesions.One of the reasons for measuring leaf lesions later than 7 days after inoculation might be environmental conditions, especially high temperatures in the period immediately after artifi cial inoculation (Hahn 2002).However, disease progress was constant and symptoms were similar with the description of this disease.Small number of lines had lesion length lower than 50% compared to susceptible control.Lines that have less than one-half of the infection percentage of highly susceptible lines could be considered as the tolerant ones according to Miller (1992) and Castano et al. (1993).Taking into consideration signifi cant correlation between lesion length and percentage of resistant plants, these lines can be considered tolerant to Sclerotinia midstalk rot.
It is known that responses to foliar and stem inoculations with mycelium are independent (Castaño et al. 1993).However, method of leaf inoculation is much closer to natural infection process than methods used for direct inoculation of stem.Moreover, methods used for direct inoculation of stem often imply artifi cial wounding in which case conclusion of some levels of tolerance might be lost.
Nevertheless, use of different methods might be needed as it is in the case with studying of white rot of head where results produced using inoculation with ascospores and mycelium proved to be complementary (Castano et al. 1993).Eleven tolerant lines were selected based on this study using method for leaf inoculation.Therefore, in order to narrow the selected lines and confirm these results, additional study using methods other than artificial inoculation of leaf will be done on all lines used in this study, with special consideration to those lines which proved to be tolerant in this study, for these lines can be used in breeding programs.

Table 1 .
In total, 36 plants per line were inoculated Successfully inoculated genotypes and their reaction to leaf inoculation with S. sclerotiorum mycelium Tabela 1. Uspešnost inokulacije i reakcija genotipova posle inokulacije sa micelijom gljive S. sclerotiorum at the budding stage, with diameter of bud approximately 5 cm