Geographic distribution and DNA-disease severity of different Rhizoctonia

Transcription

1 Geographic distribution and DNA-disease severity of different Rhizoctonia-species in wheat and corn in consideration of environmental factors and farming system G. Thomsen, J.-A. Verreet, T. Birr, H. Klink Institute of Phytopathology Christian-Albrechts-University of Kiel 1

2 - Rhizoctonia spp. - Rhizoctonia referred to a large complex group of organisms with many subspecies The pathogen causes serve symptoms and yield losses on more than 200 various plant species Damages include seedling damping-off and root rots as well as leaf-blights Isolates differ in pathogenicity, morphology, cultural and physiological characteristics Rhizoctonia spp. are soil borne, ubiquitous, necrotrophic pathogens with a wide host range and saprophytic behaviour Soil borne = wide host range; saprophytic behavoiur and the localization in soil creates a further barrier for disease control The pathogen cannot be eliminated but can be supressed to a level that doesn t cause economic losses 2

3 - Taxonomic classification - Rhizoctonia spp. Teleomorph Thanatephorus cucumeris Ceratobasidium cereale Waitea circinata Anamorph R. solani z.b. R. cerealis CAG 1 /AG-D R. zeae WAG-Z R. oryzae WAG-O Anastomosis groups (AG / CAG / WAG) AG 1 AG 13 CAG 1 CAG 7 AG-A AG-U AG 1 AG 2 AG-B Intraspecific groups (ISG) AG 1 IA AG 1 IB AG 2-2 IIIB AG-Bb Verändert nach OGOSHI, 1975; BURPEE et al., 1980; OGOSHI, 1983; OGOSHI, 1987; SNEH et al., 1998; ZENS et al.,

4 - Taxonomic classification - Within the genus Rhizoctonia three different Teleomorphs (sexual stages) are known Thanatephorus cucumeris, Ceratobasidium cereale, Waitea circinata For each Teleomorph special Anamorphs (asexual stages) are assigned Thanatephorus cucumeris Rhizoctonia solani Ceratobasidium cereale for example Rhizoctonia cerealis Waitea circinata R. zeae, R. oryzae The spezies within the anamorphs are subdivided in so called anastomosis groups (AG) R. solani is divided in 14 anastomosis groups (AG) (AG 1- AG 13 and AG BI) Within these AG s intraspecific groups (ISG) are differentiated ISG within an AG can cause different symptoms on several hosts bridging isolates (AG BI) are able to anastomoses with several AG's Number and host range of intraspecific groups (ISG) varies within the AG s (page 3, 5) 4

5 - Anastomosisgroups and their hosts - Teleomorph: Thanatephorus cucumeris Anamorph: Rhizoctonia solani AG / Host Wheat Barley Corn Sugar beet Potato Rice Soy Cotton Orchidaceae AG 1 IA X X X X X AG 1 IB X X X X AG 2-2 IIIB X X X X AG 2-2 IV X AG 3 X AG 4 X X AG 5 X X X X X AG 6 X AG 7 X X X AG 8 X X AG 9 X X X AG 10 X X AG 11 X X X X AG 12 X AG 13 X 5

6 - Taxonomic classification - There are two different anastomosis systems for the differentiation of binucleate Rhizoctonia species Japanese anastomosis systems: Species are divided in 21 anastomosis groups (AG - A to AG - U) Some of these anastomosis groups have further subgroups for example the AG-B This is subdivided in AG-Ba and AG-Bb North American anastomosis system distinguishes seven anastomosis groups CAG-1 to CAG-7 (Ceratobasidium anastomosis groups, CAG) Some of the anastomosis groups from the Japanese anastomosis system are identical to those within the North American anastomosis system in accordance of hyphal fusion 6

7 - Anastomosisgroups and their hosts - Teleomorph: Ceratobasidium cereale Anamorph: Rhizoctonia cerealis and others CAG/Host Cereals Bunch grasses (Agrostis) Peanut (Arachis) Pea (Glycine) Fleabane (Erigeron) Cheesewood (Pittosporum) Cucumber (Cucumis) CAG 1 (AG D) R. cerealis X sharp eyespot X CAG 2 (AG A) X CAG 3 (AG E) X CAG 4 (AG F) X CAG 5 (AG-R) X CAG 6 (AG E) X CAG 7 (AG-S) X Nomenclature according to the North Amerikan anastomosis system Nomenclature according to the Japanese anastomosis system 7

8 - Symptoms corn - Round to elliptical, yellow to tan or black lesions on seminal, crown, and brace roots Depending on disease severity a complete destruction of the crown roots is possible Expression of medallion lesions on shoots Root damage increases lodging, disturbed nutrient and water supply Shoot fresh matter yield reduced up to 37 %; grain yield up to 12 % (BUDDEMEYER et al., 2004) 8

9 - Symptoms wheat - R. cerealis: Symptom sharp eyespot Elliptical, light brown lesions circumscribed by a thin necrotic, darker margin on the outer leaf sheaths Rotting tissue within the lesions Caused by several AG s R. solani: bare patch : growth depression, dark green coloration of tight rolled leaves Distinct patches of stunted plants within a healthy crop MC LEOD et al., 2008 spear tips : short roots with brown lesions Disturbed nutrient and water supply Yield losses proportional to the size of the patches Caused by AG 8 HOLLAWAY et al.,

10 Faculty of Agricultural and Nutritional Science - Environmental requirements - Optimal temperature (T) R. solani = C; R. cerealis = C Soil structure = Main factor influencing the spatial distribution: Soil composition high contents of sand favored a high spread of the mycel Soil compaction high levels of mycelium in a low volume of soil Soil compaction high inoculum density high disease severity High impact of physical properties in soil: Pore size distribution, quantity of air- and waterfilled pores Influence of nutrient contents in different soils on disease severity is not clarified yet Serve symptoms under reduced tillage practice 10

11 - Objectives - Qualitative und quantitative investigations on the detection frequency and DNA-disease severity of the anastomosis groups AG 1 IA, AG 1 IB, AG 2-2 IIIB and R. cerealis in plant and soil samples Design of scecific qpcr-primers for quantification of the DNA-disease severity ( ) Identification of disease promoting or lowering environmental factors or farming systems Crop rotation physical properties in soil Assumptions for data analysis is based on studies on the influence of the pore size distribution and proportion of air- and waterfilled pores in agricultural soils Development of a weighted risk index (RI %) based on the informations about the crop rotation and the sand content at the sampled sites for the interpretation of the nationally collected data 11

12 - Experimental design - Geographic distribution and DNA-disease severity ( ) of Rhizoctonia spp. in wheat and corn Nationally Germany (D) France (F) Regional Schleswig-Holstein Experimental year 2014 Plant and soil samples 53 locations in D and F (BBCH 35 and 65) Experimental year 2015 Plant and soil samples 63 locations in D and F (BBCH 35 und 65) Experimental year 2014 Plant samples: - 10 locations corn (BBCH 65) - 7 locations wheat (BBCH 95) Soil samples: 0-5 cm cm cm (BBCH 65/95) Experimental year 2015 Plant samples: - 11 locations corn (BBCH 35 und 65) - 7 locations wheat (BBCH 35, 55, 85) Soil samples: 0-5 cm cm cm (BBCH 35 und 65/85) Experimental sites in Germany and France 2014 (left) and 2015 (right): Experimental sites wheat (left, 2014/15) and corn (right, 2014) in Schleswig-Holstein:

13 - Qualitative detection frequencies (%) of the examined Rhizoctonia-species - National locations in Germany and France, corn, experimental years 2014 und

14 - Qualitative detection frequencies (%) of the examined Rhizoctonia-species - National locations in Germany and France, experimental years 2014 und 2015 Nearly identical detection frequencies (%) of all testet Rhizoctonia-species in the experimental year 2014 Detection frequency of R. cerealis dominates with 54 % Shift towards AG 2-2 IIIB in the following year 2015, AG 1 IA couldn't be detected Germany: R. cerealis dominates clearly with a detection frequency of 65 %, decreased in 2015 (17 %) Shift towards AG 2-2 IIIB in the following year 2015 France: High dominance of AG 2-2 IIIB in 2015 (76 %) Little differences between the countries, no dominance of a special anastomosis group in Germany or France (page 13) 14

15 - Qualitative detection frequencies (%) of the examined Rhizoctonia-species - Regional locations in Schleswig-Holstein, wheat and corn, experimental years 2014 and

16 - Qualitative detection frequencies (%) of the examined Rhizoctonia-species - Regional locations in Schleswig-Holstein, experimental years 2014 and 2015 At the regional sites R. cerealis as well as AG 2-2 IIIB are of the highest detection frequency in both years 2014 and 2015 AG 1 IA and AG 1 IB are of no importance in the experimental year 2015 the AG 1 IA couldn't be detected Wheat: R. cerealis dominates clearly in both years (detection frequency > 80 %) AG 1 IA and AG 1 IB showed the same detection frequency but where only detected in 2014 or in 2015 Different sites where examined, pathogen is unable to produce asexual spores Corn: R. cerealis and AG 2-2 IIIB showed high detection frequencies in both years AG 1 IA and AG 1 IB couldn t be detected or on a very low level Pathogenicity of R. cerealis towards corn was not discribed so far 16

17 - Quantitative DNA-disease severity ( ) of the examined Rhizoctonia-species - National locations in Germany and France, experimental years 2014 and 2015 Qualitative PCR-analysis only tells whether a pathogen was detected or not There is no indication of the present amount of the pathogen and a concomitant yield relevance Quantitative real-time PCR based on the measurement of the fluorescence intensity of an DNA-intercalating reference product during a PCR (SYBR-Green) With this method the measurement of the total amount of pathogen-dna in soils (pg/g) and the pathogen-dna in relation to plant-dna ( ) in plant samples is possible Average and maximum measured amount of pathogen-dna in plant material: Regarding to the detection frequency (%) as well as the amount of pathogen-dna ( ) AG 2-2 IIIB is the most important AG compared to the other tested species within the genus Rhizoctonia followed by R. cerealis This is very interesting in view of the nearly identical detection frequencies (%) of all testet species in the experimental year 2014 AG 1 IA and AG 1 IB are detectable but in view of the amount of pathogen-dna of no importance (page 18) 17

18 - Quantitative DNA-disease severity ( ) of the examined Rhizoctonia-species - National locations in Germany and France, corn, experimental years 2014 and

19 - Disease Index (DI %) and DNA-disease severity ( ) - Depending on inoculum density on corn For validation of the measured amount of pathogen-dna within the field sampels a greenhouse experiment with all tested AG s was carried out Corn was infested with different amounts of AG-specific inoculum In BBCH 16 the plants where harvested, roots where carefully washed and rated Based on the rating a weighted disease indes (DI %) was developed Subsequently the DNA-disease severity ( ) was measured by qpcr AG 1 IA / AG 1 IB / R. cerealis: Maximum measured DNA-disease severity of 818 / 23 / 6.947, in average only 19 / 2 / 449 or no detection in the field samples Compared to the greenhouse trial there is no importance of this AG s AG 2-2 IIIB: Maximum measured DNA-disease severity of in 2014 and in 2015 In field samples measured DNA-disease severity is reproducable High damage potential of this AG can be assumed (compare page 18, 20, 21) 19

20 - Disease Index (DI %) and DNA-disease severity ( ) - Depending on inoculum density of AG 1 IA and AG 1 IB on corn, BBCH 16, greenhouse trial Maximum measured DNA-disease severity in the field samples Average of the measured DNA-disease severity in the field samples 20

21 - Disease Index (DI %) and DNA-disease severity ( ) - Depending on inoculum density of AG 2-2 IIIB and R. cerealis on corn, BBCH 16, greenhouse trial Maximum measured DNA-disease severity in the field samples Average of the measured DNA-disease severity in the field samples 21

22 - Determination of the risk index (RI %) - Classification of the locations regarding to,crop rotation and,sand content For the interpretation of the measured DNA-disease severities a weighted risk index (RI %) was developed The previous crops of the sampled sites where divided in three classes: 1. crop rotation wide (CR wide) 2014: no sugar beet or corn as previous crop; 2015: 3 years no sugar beet or corn 2. crop rotation limited (CR lim.) up to 2 years sugar and beet corn, corn and sugar beet or corn and corn 3. monoculture more than 3 years corn as previous crop on the sampled site In 2015 the sand content was used for the assesment of favoured soil physical properties based on the informations given by the supervisor of the sampled sites The sand content of the soil was also divided in three classes: 1. sand content low (SC low) 2. sand content medium (SC medium) 3. sand content high (SC high) (page 23) 22

23 - Determination of the risk index (RI %) - Classification of the locations regarding to,crop rotation and,sand content Previous crop 2014 Previous crop 2015 Sand content Jahre Wide rotation ( CR wide ) Limited rotation ( CR lim. ) 2 Jahre Monoculture 3 Jahre Soil types of fine soil, FAO and World Reference Base (WRB), changed according to BLUME et al., 2010 sand content medium sand content high 23

24 Proportion fraction (%) Agrar- und Ernährungswissenschaftliche Fakultät - Bases for the definition of the factor sand content - Factor sand content is an auxiliary quantity Proportion of coarse pores builds up with increasing sand content Proportion of fine pores is greater in fine-grained soils In sandy soils, the proportion of coarse pores are dominating Pore size is critical for root growth and microbial activity Root hairs penetrate only coarse pores; Fungi / bacteria also live in medium sized pores Fine pores are inaccessible to microorganisms clay silt sand fine medium coarse fine medium coarse fine medium coarse Pore vol. (%) Coarse pores (%) Medium pores (%) Fine pores (%) sand 46 ± ±10 7 ± 5 5 ± 3 silt 47 ±9 15 ± ± 7 15 ± 5 clay 50 ±15 8 ± 5 10 ± 5 35 ± 10 Grain size Table: Pore volume and pore size ranges of the total pore volume of mineral and organic soils Figure: Cumulative curve of fine soils, sand (Ss), loess (Ut), boulder-clay (Ls) and clay (Tu), changed according to BLUME et al.,

25 - Derivation of the RI (%) for the factors crop rotation and sand content - The sampled sites where classified independently in view of the crop rotation and sand content The average of the DNA-disease severity ( ) for each analysed pathogen was calculated and divided in three classes 1. No detection 2. Below the average 3. Above average Subsequently these classes of DNA-disease severity ( ) where weighted No detection = 0 points Below the average = 1 point Above the average = 2 points Followed by a counting of the number of sites in the three classes of the two factors depending on the level of DNA-disease severity ( ) After assigning the number of sites to the corresponding class of measured DNA-disease severity ( ) of the regarded AG within each factor (crop rotation or sand content) the risk index (RI %) was calculated (page 26) 25

26 - Derivation of the RI (%) for the factors crop rotation and sand content - 26

27 27 Agrar- und Ernährungswissenschaftliche Fakultät - Calculation risk index (RI %) - For example,crop rotation and sand content, AG 2-2 IIIB in Germany and France experimental year 2015 No detection Below the average Above the average Locations in Germany and France :

28 - Influence of,crop rotation on the RI (%) of AG 1 IA and AG 1 IB - National locations in Germany and France, experimental year 2014, (n=53) Small increase of RI (%) in view of AG 1 IA in corn when grown in monoculture Higher risk of infestation with AG 1 IB in wide crop rotations, only latent infection of corn in limited crop rotation or monoculture RI (%) depends on host plant specific coverage / proportion of host plants within crop rotation 28

29 - Influence of the,crop rotation on the RI (%) of AG 2-2 IIIB and R. cerealis - National locations in Germany and France, experimental year 2014, (n=53) Strong increase of RI (%) in view of AG 2-2 IIIB in corn when grown in monoculture Higher risk of infestation with R. cerealis in wide or limited crop rotations, host plant specificity is stronger towards cereals which are grown in a wide crop rotation, at monoculture sites only a latent infection on corn is detectable, starving until cereals are grown on the same site again Corn is of no relevance in view of loosening the crop rotation for disease control of R. cerealis on cereals RI (%) depends on host plant specific coverage / proportion of host plants within crop rotation 29

30 - DNA-disease severity ( ) AG 2-2 IIIB / R. cerealis in plant material - Regional locations Schleswig-Holstein These results can be confirmed with the results from the regional analysed sites in Schleswig- Holstein Higher detection frequencies (%) and DNA-disease severities ( ) of R. cerealis in corn where measured in plant material from sampled sites if corn was grown in crop rotations with cereals AG 2-2 IIIB was of high detection frequencies (%) and DNA-disease severities ( ) if corn was grown in monoculture (page 31) The host plant specificity of R. cerealis is higher towards cereals Within the regional project additionally wheat samples where analysed In 2015 three growth stages where tested R. cerealis showed higher DNA-disease severities ( ) in wheat samples and the pathogen was detected at early stages of development As well as in the samples from the national locations R. cerealis showed only little DNA-disease severities ( ) in corn samples but high detection frequencies (%) (page 32) This effect was observed in both experimental years (page 33) 30

31 - DNA-disease severity ( ) AG 2-2 IIIB / R. cerealis in plant material - Regional locations Schleswig-Holstein, Corn BBCH 65, experimental year

32 - DNA-disease severity ( ) of R. cerealis in plant material - Regional locations Schleswig-Holstein, wheat BBCH 35, 55 and 85; corn BBCH 35 and 65 experimental year

33 - DNA-disease severity ( ) R. cerealis in plant material in S.-H. - Wheat (W) BBCH 85; corn (C) BBCH 65, experimental years 2014 and

34 - Influence of,crop rotation and,sand content on RI (%) of AG 2-2 IIIB - National locations in Germany and France, experimental year 2015, (n=63) The results for the influence of crop rotation on RI (%) of AG 2-2 IIIB could also be confirmed in 2015 With narrowing up the crop rotation from a wide one to monoculture the RI (%) of AG 2-2 IIIB increased But also the sand content of the sampled sites showed an effect on RI (%) With a change of by the pathogen favoured soil physical properties based on a rising sand content (higher amount of coarse pores which influenced fungal growth) RI (%) increases This effect can also be shown in view of the influence of the factor sand content by a differentiation of the sampled sites in the classes crop rotation (no differentiation between wide or limited rotations) and monoculture If corn was grown in crop rotation RI (%) is on a lower level if the sand content of the sampled sites is low With increasing sand content RI (%) increases because of more favorable soil physical conditions Anyway, if corn was grown in monoculture RI (%) is on a higher level But also in case of monoculture RI (%) is increasing again with a rising sand content The factor crop rotation superimposes the factor sand content (page 35) For the pathogen the availability of a suitable host plant is more important If a host plant is absent, the RI (%) increases with a change of soil physical properties towards more favourable conditions for fungal growth 34

35 - Influence of,crop rotation and,sand content on RI (%) of AG 2-2 IIIB - National locations in Germany and France, experimental year 2015, (n=63) 35

36 - Detection frequencies (%) of all tested Rhizoctonia-species in soil samples - National locations in Germany and France, experimental years 2014 and 2015 Low detection frequencies in soil samples 71 % of the locations with a positive pathogen detection of AG 2-2 IIIB in soil samples the pathogen was also detected in plant material in 2014 At most locations with positive detection corn was grown in monoculture 36

37 - Pathogen-DNA (pg/g) of all tested Rhizoctonia-species in soil samples - National locations in Germany and France, experimental years 2014 and 2015 Overall the detection frequencies (%) of the tested Rhizoctonia-species in soil samples are on a low level The highest detection frequencies (%) are observed for AG 2-2 IIIB with 26 % in 2014 and 25 % in 2015 AG 1 IA could not be detected in both experimental years With division of the sampled sites in view of the geographic localization in Germany and France, no relevant differences in the detection frequencies (%) of the pathogens are detectable Little differences in the measured amounts of pathogen-dna (pg/g) with exception of AG 2-2 IIIB in the experimental year

38 - Detection of the tested Rhizoctonia-species in soil samples - Regional locations in Schleswig-Holstein, experimental years 2014 and 2015 At the regional sites the soil samples where divided in three soil depths (0-5, 10-15, cm) In 2014 highest amounts of pathogen-dna of AG 2-2 IIIB where measured in the soil depth of cm A detection of this anastomosis group was only possible at sites where corn was grown (page 39) R. cerealis was detected at only one site where corn was grown but again the soil depth of cm Highest detection frequencies (%) and amount of pathogen-dna (pg/g) where measured in the soil depth of cm and at sites where wheat was grown (page 40) In the following year a detection of the pathogen in the soil depth of cm and an increase of pathogen-dna within the soil samples was supposed A confirmation of the results from the previous year 2014 was not possible (page 41) AG 1 IA and AG 1 IB where not detected in both years 38

39 - Detection of AG 2-2 IIIB in soil samples - Regional locations in Schleswig-Holstein, experimental year 2014 Soil depth 0-5, und cm 39

40 - Detection of R. cerealis in soil samples - Regional locations in Schleswig-Holstein, experimental year 2014 Soil depth 0-5, und cm 40

41 - Detection of AG 2-2 IIIB in soil samples - Regional locations corn in Schleswig-Holstein, experimental years 2015 Soil depth 0-5, und cm, BBCH 35 and 65 41

42 - Summary - In both years at least one of the four investigated species could be detected in 83 % of the locations surveyed in Germany and France In Schleswig-Holstein, 100 % of the sampled sites achieved positive pathogen detection in both years Qualitatively and quantitatively, the AG 2-2 IIIB and R. cerealis dominate depending on the plant specific degree of coverage (proportion of host plants within a crop rotation/monoculture) and the studied culture A limited crop rotation up to monoculture greatly increases the RI (%) of AG 2-2 IIIB Within monoculture, the RI (%) increases again by an increase in the sand content of the soils of the experimental sites Factor,crop rotation superimposes factor,sand content R. cerealis can be detected in higher DNA-disease severities ( ) in wheat, in corn mainly in wide to limited crop rotations The higher the host plant specificity, the better the results can be expressed as a RI (%) Low detection frequencies in soil samples, strongly varying pathogen-dna quantities 42