3 Results
3.4 Experiments under controlled conditions
3.5.2 Monitoring spore dispersal by rain and snowfalls
In the present study, rain and snow samples were collected at several kilometres distance from barley fields and about 20m above the ground. DNA extractions were performed from rain and snow samples, which were used for qualitative PCR analysis. Among twenty three samples, which were collected during October 2008 to January 2009, 6 (23%) were found to be positive for Rcc (Fig. 32). These results indicate that Rcc was detectable during late autumn and winter months at larger distances from fields and in higher elevation above ground. We therefore assumed that Rcc inoculum is widespread also in the cooler season and may spread over large distances via the atmosphere and in rain water or snow.
1 2 3 4 5 6 7 8
73 Figure 32. Gel electrophoresis of Rcc DNA in rain and snow samples which were detected by PCR. Lanes: 100-bp DNA ladder Plus (Fermentas); 1) negative control; 2) positive control (pure Rcc DNA) 3, 4, 5, 6 and 8) rain samples which were gathered from 22.10.2008 to 05.01.2009; 7) snow sample which was gathered at 26.11.2008
3.5.3 Resistance screening under field conditions
Although temperature, rainfall, and relative humidity were different in July 2009 and July 2010, there was a good agreement among the screening results. There were significant differences (p≤0.05) among different spring barley genotypes in different two years under field conditions. Symptom development in the field occurred relatively late in the plant development almost when the plants were at flowering stage. In both years, percentage of disease symptoms for the most genotypes was less than 10% at early growth stage (61-65). Due to the warm conditions in 2010, the disease level was relatively lower in this year in comparison with 2009 (Fig. 33).
20 40 60 80 100
%Necrotic leaf area
Median; 25%-75%; Min-Max
Figure 33. Disease severity (%) on F-1 leaves of forty different spring barley genotypes at early and late growth stages in two different years (2009 and 2010). Data show Box-Whisker-Plots with median values. Borders of boxes represent 25% and 75% quartiles, thus boxes contain 50% of observed values. Boxes represent 25-75% of the data and whiskers contain minimum and maximum values.
For field scoring in 2009, first at GS 61-65, 50 plants per each replicate (in total 150 plants) were harvested and three upper leaves (flag leaf, F-1 and F-2) of each plant were scored for RLS by visually estimating the percentage of necrotic leaf area. In all genotypes, symptoms first become visible on leaf F-2 with maximum amount of 23.63% (±09.42) for Victoriana and 0.0% for IPZ 24727. Disease symptoms on F-1 ranged from 0.0% for IPZ 24727 to 15.33% (±13.57) for Signiora. No visible symptoms were observed on flag leaves. At GS 73-75, scoring of disease symptoms was repeated as like in GS 61-65. Symptoms were clearly visible on all leaves at this time point (Table 12) except for cultivar Gaute which was infected strongly by powdery mildew which made it impossible to score the Rcc disease symptoms.
Table 12. Susceptibility of different spring barley cultivars to RLS in the field trial in 2009 under natural infection conditions. At growth stage 73-75, the three upper leaves were scored by estimating the percentage of necrotic leaf area; data show the mean of 150 (3*50) replicates with standard deviations (in brackets)
Spring barley % Necrotic leaf area (±SD)1
Sebastian 8.00 (±03.68) abcdefg 74.19 (±06.67) ghijk 86.33 (±04.42) ghijklmn Signiora 33.67 (±18.07) pqrst 71.25 (±09.35) ghijk 89.00 (±04.31) jklmno Styx 12.47 (±14.26) defghij 65.67 (±11.78) cdef 88.00 (±11.77) ijklmno Umberella 2.93 (±02.74) abc 59.19 (±06.21) c 77.00 (±08.19) ef Varberg 5.53 (±04.17) abcd 43.86 (±13.65) b 61.67 (±17.39) c Victoriana 14.80 (±13.41) efghijkl 77.19 (±17.90) ijklmno 86.67 (±12.49) ghijklmn Waldemar 18.33 (±08.16) ijklmn 78.53 (±08.12) jklmno 86.00 (±04.71) ghijklmn
1 Percentage of necrotic leaf area due to RLS on three different leaves at GS 73-75. Values with different letter within the same column indicate significant differences (p ≤ 0.05) between the genotypes, calculated by the LSD-Fisher test
In 2010, due to strong correlation between the results of disease symptoms on the flag leaf and F-1 (rs=0.7899; p=0.0000) and F-1 to F-2 (rs=0.8372; p=0.00001) at GS 73-75 in 2009, the percentage of necrotic leaf area was only estimated on F-1 leaf. At different growth stages (61-65, 65-69, 71-73, and 73-75), fifty F-1 leaves were harvested randomly per each replicate (in total 150 plants) and scored for RLS by visually estimating the percentage of necrotic leaf area (Table 13).
Table 13. Susceptibility of different spring barley cultivars to RLS at field trial in 2010 in Lenglern/Germany under natural infection conditions. Leaf F-1 was scored by estimating the percentage of necrotic leaf area at different growth stages; data are means of 150 (3*50) replicates with standard deviations (in brackets)
% Necrotic leaf area (±SD)1
Barley
78
1 Percentage of necrotic leaf area due to RLS on leaf F-1 at different growth stages. Values with different letter within the same column indicate significant differences (p ≤ 0.05) between the genotypes at one time point, calculated by the LSD-Fisher test
According to table 13 the development of disease symptoms in 2010 was low until GS 71-73. In that year, at GS 61-65, the percentage of disease symptoms on F-1 leaves ranged from 0.20 (±0.41) for IPZ 24727 to 3.93 (±3.07) for Waldemar. Disease development increased gradually until GS 71-73 with a minimum amount of 2.10%
(±1.95) for IPZ 24727 and a maximum amount of 45.67% (±6.66) for Braemar.
Favourable conditions for pathogen development between GS 71-73 and GS 73-75 resulted in a dramatic increase in disease symptoms in most barley genotypes, which ranged from 5.10% (±03.23) for IPZ 24727 to 82.33% (±05.68) for Quench.
Although the cultivar IPZ 24727 showed the strongest resistance reaction to Rcc on F-1 at GS 73-75 with the disease severity levels of 18.33% in 2009 and of 5.10% in 2010, the other cultivars were more or less susceptible. In 2009 the most susceptible cultivars were Barke (90.86%), Hatifa (90.53%), and Conchita (87.86%). Spring barley cultivars Quench (82.33%), Signiora (81.17%), and Scarlett (80.07%) were the most susceptible genotypes in 2010. Dry conditions in 2010 resulted in a limited Rcc epidemic. Rcc is favoured by high humidity conditions and average temperatures of 15 to 20 °C. Meteorological data indicated that temperature was less and relative humidity was much higher during disease development in July 2009 compared to July 2010 (Fig. 34).
Figure 34. Comparison of average day temperature (°C) and relative humidity (%) in July 2009 and July 2010 during Rcc infection on the field trial. White columns indicated average day temperature in 2009, which were much lower than black columns (average day temperature in 2010) especially from 1st to 15th of July during major disease development.
The white symbols indicate percentage of relative humidity in 2009, which was higher than 2010 (black symbols). Data were recorded by the German Weather Service (DWD), Göttingen station.
The analyses of variance for different genotypes in each year at late growth stage (73-75) and the comparison of each mean value of disease symptoms for each genotype in two years are given in Table 14.
Table 14. Comparison of susceptibility of different spring barley cultivars to RLS in field trials under natural infection conditions in two different years (2009 and 2010) in Lenglern/Germany. Leaf F-1 was scored by estimating the percentage of necrotic leaf area at growth stages 73-75; data are means of 150 (3*50) replicates with standard deviations (in brackets)
Barley
Barke 90.86 (±12.27) p 73.83 (±08.96) nop 0.005769
1 Percentage of necrotic leaf area due to RLS on leaf F-1 in growth stage 73-75. Values with different letter within the same column indicate significant differences (p ≤ 0.05) between the genotypes in one year, calculated by the LSD-Fisher test
2 t-tests between the mean values of each genotype in two different years; significant differences at p ≤ 0.05
Although different results were scored for the field trials in two consecutive years, a significant correlation (rs=0.419) was observed between field experiments in two different years at growth stage 73-75 (Fig. 35).
Figure 35. Correlation between disease severity (% necrotic leaf area) in field experiment 2009 and field experiment 2010, both at Lenglern/Germany. rs: Spearman's rank correlation coefficient, p: p-value ≤0.05
Spore trap: In the year 2010, two spore traps, each containing 4 Petri dishes, were installed in both left and right side of the field trial. According to Fig. 36, the mean number of Rcc colonies remained more or less constant from beginning to middle of
0 20 40 60 80 100
% Disease severity (field trial 2010) 0
20 40 60 80 100
% Disease severity (field trial 2009)
y= 34.311+ 0.5879*x; p= 0.0414; rs=0.419
0 5 10 15 20 25 30 35
01.06.2010 04.06.2010 07.06.2010 10.06.2010 13.06.2010 16.06.2010 19.06.2010 22.06.2010 25.06.2010 28.06.2010 01.07.2010 04.07.2010 07.07.2010 10.07.2010 13.07.2010 16.07.2010 19.07.2010 22.07.2010 25.07.2010 28.07.2010
Date Number of Rcc colony in the spore trap Spore trap A
Spore trap B
June in both spore traps. Surprisingly, the mean numbers of colonies on spore trap B showed highly significant differences (p≤0.05) in comparison to spore trap A during the middle to almost late June. This great peak was detected a week before the first symptoms were visible on the field and plants were at growth stage 55-59. Field observation showed that the main source of this primary inoculum came from a strongly Rcc infected winter barley field which was located close to our field trial and about 30m away from spore trap B. The second major peak was observed almost four weeks later when the plants were at growth stage 75/79 and completely were infected by Rcc.
Figure 36. Average density of Rcc spores in spore traps, which were installed on both sides of a field trial from 1st of June until 31st of July 2010; Lenglern, Lower Saxony, Germany.
Weather data during the experiments indicated that environmental conditions did not have strong effect on spore deposition (Fig. 37).
83
24727 Thule Nymfe isanne Power oriana Marthe igniora Barke uench
ng fungal DNA in 1 gr dreid leaves
GS 61-65