Characterization of phosphate solubilisation and uptake capacity by root fungal endophytes

Size: px

Start display at page:

Download "Characterization of phosphate solubilisation and uptake capacity by root fungal endophytes"

Mae Sullivan
5 years ago
Views:

fungal endophytes Diana Rocio Andrade-Linares Benard Ngwene Philipp Franken COST

1 Leibniz-Institut für Gemüse- und Zierpflanzenbau Großbeeren & Erfurt Leibniz Gemeinschaft Characterization of phosphate solubilisation and uptake capacity by root fungal endophytes Diana Rocio Andrade-Linares Benard Ngwene Philipp Franken COST FA113: Endophytes in Biotechnology and Agriculture 14-16/11/12, S. Michele all Adige, Trento, Italy.

2 Facultative symbiontes in roots Piriformospora indica Trichoderma harzianum Dark septate endophytes DSE INTR

3 Fungal endophytes Contribution to mineral element uptake and promotion of plant growth AM Association T3 RE association Intra- & extraradical structures Root cortex: IRM Soil: ERM E27 Root cortex: IRM Soil: ERM? Intraradical structures Andrade-Linares et al 211 (Mycology) N E48

4 Research focus Fungal endophytes To evaluate phosphate solubilization of organic and inorganic phosphate sources and the potential production of phosphatases in liquid medium To test the capability of Piriformospora indica to use organic nitrogen in vitro system To evaluate inorganic phosphate transfer, plant growth promotion in tomato plants. To evalute ERM growth of P. indica into root inaccessible soil compartments To analyze the interaction between P. indica and Glomus intraradicis in the symbiosis with tomato plants

5 General methodology 1. Phosphate solubilization Lecithin Phytin Pikovskaya medium Rock Phosphate Agar Different concentrations Liquid culture Available Phosphorous and enzimatic activity DSE48 L. orchidicola Protease activity in vitro on Casein source P. indica 3. Phosphorous uptake in pot experiments

1. Phosphate solubilization 2. Available Phosphorous and enzimatic activity Lecithin Phytin Pikovskaya agar Liquid culture KH 2 PO 4 (499 mg/l P) Tri-calcium phosphate (TCP, Ca 3 O 8 P 2 ;.

6 1. Phosphate solubilization 2. Available Phosphorous and enzimatic activity Lecithin Phytin Pikovskaya agar Liquid culture KH 2 PO 4 (499 mg/l P) Tri-calcium phosphate (TCP, Ca 3 O 8 P 2 ;.25%) Rock Phosphate (RP, P 2 O 5 ;.347% ) Lecithin Phytin Phosphate concentrations: 5, 2, 1, 5, mg/l Growth analysis and hydrolisis during 3 days of incubation Sampling each 5 days P mg/l ph Shaker at 25 o C Enzymatic activity

7 Assimilation and solubilization of different P- sources Growth in Lecithin Growth in Phytin DSE48 Colony diameter (cm) 5mg/L 2mg/L 1mg/L 5mg/L mg/ml 4 p =, Incubation days at 24 o C Colony diameter (cm) 4 2 5mg/L 2mg/L 1mg/L 5mg/L mg/ml p=, Incubation days at 24 o C 25 L. o 135 Colony diameter (cm) 5mg/L 2mg/L 1mg/L 5mg/L mg/ml 6 p =, Incubation days at 24 o C 25 Colony diameter (cm) 5mg/L 2mg/L 1mg/L 5mg/L mg/ml 4 p =, Incubation days at 24 o C P. indica Colony diameter (cm) mg/L 2mg/L 1mg/L 5mg/L mg/ml p =, Incubation days at 24 o C Colony diameter (cm) 1 5 5mg/L 2mg/L 1mg/L 5mg/L mg/ml p =, Incubation days at 24 o C

8 Available Phosphorous and Enzimatic activity Lecithin 5 mg/l Phytin 15 mg/l Phytin L.o135 Phytin P. indica Phytin control DSE P mg.l incubation days L.o135 P. indica control DSE48 Phy Lo135 Phy P. indica Phy DSE48 Phy contro l(-) 7,,47 6, Acid Phosphatases (U/ml) ph,37 5, 4,,27 3,,17 2,,7 1,, -, Incubation incubation days at 24 o C

9 Assimilation and solubilization of different P- sources Pikovaskaya agar with different concentrations of Ca 3 (PO 4 ) 2 = (Ca 3 -P) DSE48 Colony diameter (cm) 5mg/L 2mg/L 1mg/L 5mg/L mg/ml 1 p =, Incubation days at 24 o C Halo hydrolisys (cm) mg/L 2mg/L 1mg/L 5mg/L p =,65 B Incubation days at 24 o C L. o 135 Colony diameter (cm) 1 5mg/L 2mg/L 1mg/L 5mg/L mg/ml 5 p =, Incubation days at 24 o C Halo hydrolysis (cm) 1 5 5mg/L 2mg/L 1mg/L 5mg/L p =,134 A Incubation days at 24 o C P. indica Colony diameter (cm) 5mg/L 2mg/L 1mg/L 5mg/L mg/ml 1 p =, Incubation days at 24 o C

10 Available Phosphorous and Enzimatic activity Ca 3 -P 5 mg/l Ca 3 -P 15 mg/l 3Ca -P L.o135 3Ca -P E48 3Ca -P P. indica 3Ca -P control 3Ca -P L.o135 3Ca -P control 3Ca -P P. indica DSE48 P mg.l P mg.l incubation days incubation days ph L.o135 ph P. indica ph control 3Ca -P E48 L.o135 P. indica control DSE48 ph 7, 6, 5, 4, 3, ph 7, 6, 5, 4, 3, 2, 1,, , 1,, incubation days incubation days

11 Available Phosphorous and Enzimatic activity Ca3-P 5 mg/l Ca3-P 15 mg/l KHPO 135 KHPO Piri 3Ca 135 3Ca Piri RP 135 RP Piri 3CaP DSE48 3CaP L.o135 3CaP P. indica 3CaP control (-),24,72 Acid phosphatases (U/ml),19,14,9,4 -, incubation days Acid phosphatases (U/ml),62,52,42,32,22,12,2 -, Incubation days

2. Protease activity a Diameter of colony [cm] 6 5 4 3 2 1 a a b c a (Brunel, J.

12 2. Protease activity a Diameter of colony [cm] a a b c a (Brunel, J. et al. 212) B a A colony t =,814 c =, tc =,2148 hydrolysis t =,1 Ara + Piri Piri Ara + Piri Piri Ara + Piri Piri MS MS Cas MS Cas Milk Treatment Conditions b A. thaliana MS Cas Milk A. thaliana + P. indica Proteolysis indicator P. indica

13 conclusions Conclusions Lecithin inhibits growth of the DSE48 and P. indica, while L. orchidicola 135 is able to growth up to a concentration of 5 mg/l, however the release of P i is low (2mg/L) and after 2 days of incubation. The endophytes growth in high concentration of Phytin, specially P. indica, however it did not release P i and not present enzymatic activity in contrast to L. orchidicola 135. The fungal endophytes grow better at concentrations higher than 1 mg/l in Pikovskaya (Ca 3 -P) agar but the solubilization was less evident at 5 mg/l (PO 4 ) 2. P. indica does not produce hydrolysis halo in comparison to the DSE in Ca 3 - P but it is able to release P i in liquid medium and seems to be more efficient at higher concentration (5 mg/l) in contrast to L. orchidicola 135. However P. indica does not have extracellular phosphatase activity. P. indica does produce hydrolysis halo in casein agar which indicate the production of proteases which seems to be regulated by the host plant.

14 General methodology 1. Phosphate solubilization Lecithin Phytin Pikovskaya medium Rock Phosphate Agar Different concentrations Liquid culture Available Phosphorous and enzimatic activity DSE48 L. orchidicola Protease activity in vitro on Casein source P. indica 3. Phosphorous uptake in pot experiments

3. Phosphorous uptake: Pi content in plant

extraction Barley plants Inoculation Trap

Optimal Pi 2mg Growth parameters P in plant

15 3. Phosphorous uptake: Pi content in plant Root colonization Extraradical Mycelium 1 phase 2 phase P. indica Glomus P+Glo Tomato plants PCR DNA extraction Barley plants Inoculation Trap soil 8 weeks + Compartment Low Pi 5mg Optimal Pi 2mg Growth parameters P in plant Endophytic growth DNA extraction PCR 7 weeks + Hoadglan -Pi Endophytic colonization

indica P. indica + Glomus P. Indica + G.

16 Tomato colonized roots (frequency and intensity),7 a Frequency P. indica Colonization in tomato roots,6,5,4,3,2,1 b A A a A ab A %, P. indica P. indica + Glomus P. Indica + G. intraradicis Colonization in tomato roots 1,,9,8,7,6,5,4,3,2,1 a A a A a AB b B Frequency %, Glomus Glomus + P. indica G. intraradicis

17 Shoot dry weight after harvest (g per plant) Shoot dry weight (g/plant) P +P -P +P -P +P -P +P -P +P Effect of inoculation treatment Control Piri Control Glomus P. indica Glomus P. indica + Glomus

18 Shoot P uptake after harvest (mg per plant) 1 Shoot P uptake (mg per plant) Inoculum: p <,1 Root length colonized (%) P +P -P +P Glomus Glomus + P. indica Effect of inoculation treatment -P +P -P +P -P +P -P +P -P +P Control Piri Control Glomus P. indica Glomus P. indica + Glomus

Detection of extraradical mycelium of P.

19 Detection of extraradical mycelium of P. indica Soil of compartments Soil for trap pots with barley P. Indica in: Pots (C) ITS1-4 Piritef + Piritef GAPDH (C) + (C) + Primer pair for the translation elongation factor 1 α gene (TEF) : Piritef Primer pair for the glyceraldehyde-3-phosphate dehydrogenase gen (GAPDH) P. indica and G. intraradicis in Compartment (C)

20 Colonization of trap plant roots 2 P. indica spores and hyphae attaching root hair of barley plant Barley plants Trap soil 7 weeks Endophytic colonization Percent of colonization in barley roots P. indica spores inside root hair of barley plant without plant with plant Control (+) frequency Intensity

21 conclusions Conclusions Tomato root plants were colonized at the same time by P. indica and G. intraradicis. This colonization seems to be lower for P. indica than for G. intraradicis. However the AMF colonization was negatively affected in the combination treatment (Piri + Glomus) at high concentration of P i in the compartment. The inoculation with the endophyte P. indica showed a significant effect on plant dry weight as well as on P uptake and this effect is higher with the presence of the AMF in roots. P. indica is able to produce ERM which was detected by PCR in soil compartments and was also recovered in the roots of trap plants.

22 Phosphorous and nitrogen uptake from organic sources and interaction witn AMF. Quantification of extraradical mycelium and root colonization at saprophytic and endophytec stages in soil. Regulation of phosphate transportes Influence of endohyphal bacteria on fungal and plant nutrition.

23 Institute of Vegetable and Ornamental Crops Grossbeeren and Erfurt e.v. Many thanks to: Dr. Bernard Ngwene Prof. Eckhard George Prof. Philipp Franken IGZ Thanks for your attention

NEW MAN INTERNATIONAL JOURNAL OF MULTIDISCIPLINARY STUDIES (ISSN: ) Kolkar M.V., Bhosle P.K., Deo M.S., Dr. Bhutada S.A.

NEW MAN INTERNATIONAL JOURNAL OF MULTIDISCIPLINARY STUDIES (ISSN: ) Kolkar M.V., Bhosle P.K., Deo M.S., Dr. Bhutada S.A. 6 PHOSPHATE SOLUBILIZING BACTERIA AND THEIR ROLE IN PLANTS Kolkar M.V., Bhosle P.K., Deo M.S., Dr. Bhutada S.A. Abstract: - Phosphorous is major plant growth limiting nutrient, though P is abundant in